Peptidomimetic macrocycles

ABSTRACT

The present invention provides peptidomimetic macrocycles capable of modulating growth hormone levels and methods of using such macrocycles for the treatment of disease.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.14/750,649 filed Jun. 25, 2015, issued as U.S. Pat. No. 9,522,947 onDec. 20, 2016, which is a continuation of U.S. Non-Provisionalapplication Ser. No. 13/655,378 filed Oct. 18, 2012, issued as U.S. Pat.No. 9,096,684 on Aug. 4, 2015, which application claims the prioritybenefit of U.S. Provisional Application Ser. No. 61/548,690 filed Oct.18, 2011, the content of each of which is hereby incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 24, 2016, isnamed 35224-769_302_SL.txt and is 225,340 bytes in size.

BACKGROUND OF THE INVENTION

Human GHRH (Growth Hormone-Releasing Hormone) is a 44-amino-acid peptidewhose full biological activity resides in its first 29 amino acids(“GHRH 1-29”). GHRH binds to the GHRH receptor and stimulates pulsatileGH [Growth Hormone] secretion, and with this mechanism of action GHRHrepresents an alternative to GH therapy in patients with an intactpituitary that may minimize the side effects associated with long-termGH administration. Because the quantity of GH release induced by GHRH islimited by IGF-1 levels, which exert a negative feedback effect, therisk of side effects associated with excessive GH secretion may also belower with GHRH therapy than with GH therapy. In addition, treatmentwith GHRH may result in the pituitary secretion of a broader set of GHproteins, and not just the 22-kDa form provided by recombinant human GH,which may also have beneficial effects. Clinically, GHRH has been shownto be safe and effective in increasing GH levels in adults and children,and the growth-promoting effect of GHRH is correlated with the dose andfrequency of administration. However, the half-life of GHRH afterintravenous injection is only 10-12 min, which has significantly limitedits use as a therapeutic agent. Thus there is a clinical need foranalogs of GHRH that possess extended half-life in vivo that couldprovide greater therapeutic benefit with an improved (less frequent)dosing regimen.

SUMMARY OF THE INVENTION

The present invention provides GHRH-derived peptidomimetic macrocyclesthat are designed to possess improved pharmaceutical properties relativeto GHRH. These improved properties include enhanced chemical stability,extended in vivo half-life, increased potency and reducedimmunogenicity. These peptidomimetic macrocycles are useful to increasecirculating levels of GH as a treatment for muscle wasting diseases,lipodystrophies, growth hormone disorders, gastroparesis/short bowelsyndrome, and other conditions for which an increase in GH would providetherapeutic benefit.

Described below are stably cross-linked peptides derived from the GHRHpeptide. These cross-linked peptides contain at least two modified aminoacids that together form an intramolecular cross-link that can help tostabilize the alpha-helical secondary structures of a portion of GHRHthat is thought to be important for agonist activity at the GHRHreceptor. Relative to the amino acid sequence of the wild-type peptide,any amino acid which is not essential to the growth-hormone releasingactivity of the peptide may be replaced with any other amino acids,while amino acids which are essential to the growth-hormone releasingactivity of the peptide may be replaced only with amino acid analogswhich do not substantially decrease said activity.

Accordingly, a cross-linked polypeptide described herein can haveimproved biological activity relative to a corresponding polypeptidethat is not cross-linked. Without being bound by theory, the GHRHpeptidomimetic macrocycles are thought to activate the GHRH receptor,thereby stimulating production and release of growth hormone, which canincrease lean muscle mass or reduce adipose tissue (such as abdominaladipose tissue). For example, adipose tissue can be reduced in subjectssuffering from obesity, including abdominal obesity. The GHRHpeptidomimetic macrocycles described herein can be used therapeutically,for example, to treat muscle wasting diseases that include anorexias,cachexias (such as cancer cachexia, chronic heart failure cachexia,chronic obstructive pulmonary disease cachexia, rheumatoid arthritiscachexia) and sarcopenias, to treat lipodystrophies that include HIVlipodystrophy, to treat growth hormone disorders that include adult andpediatric growth hormone deficiencies, or to treat gastroparesis orshort bowel syndrome. Pediatric growth hormone deficiency may be, forexample, linked with or associated to idiopathic short stature, SGA(infant small for gestational age), chronic kidney disease, Prader-Willisyndrome Turner syndrome, short stature homeobox (SHOX) gene deficiency,or primary IGF-1 deficiency.

In one aspect, the present invention provides a peptidomimeticmacrocycle comprising an amino acid sequence which is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identicalto GHRH 1-44, GHRH 1-29 and/or to an amino acid sequence chosen from thegroup consisting of the amino acid sequences in Table 1, 2 or 4.Alternatively, an amino acid sequence of said peptidomimetic macrocycleis chosen from the group consisting of the amino acid sequences in Table1, 2 or 4. The peptidomimetic macrocycle may comprise one, two, three,four, five or more macrocycle-forming linkers, wherein eachmacrocycle-forming linker connects one amino acid to another amino acidwithin the peptidomimetic macrocycle. For example, a peptidomimeticmacrocycle comprises at least two macrocycle-forming linkers wherein thefirst of said at least two macrocycle-forming linkers connects a firstamino acid to a second amino acid, and the second of said at least twomacrocycle-forming linkers connects a third amino acid to a fourth aminoacid. In some embodiments, the peptidomimetic macrocycle comprisesexactly two macrocycle-forming linkers. In other embodiments, thepeptidomimetic macrocycle comprises exactly one macrocycle-forminglinker.

Macrocycle-forming linkers connect any two amino acids which can becrosslinked without impairing the activity of the peptidomimeticmacrocycle. In some embodiments, a macrocycle-forming linker connectsone of the following pairs of amino acids (numbered with reference toany sequences aligned to GHRH 1-29): 4 and 8; 5 and 12; 8 and 12; 8 and15; 9 and 16; 12 and 16; 12 and 19; 15 and 22; 18 and 25; 21 and 25; 21and 28; 22 and 29; 25 and 29. For example, a macrocycle-forming linkersconnects of the following pairs of amino acids: 4 and 8; 5 and 12; 12and 19; 15 and 22; 18 and 25; 21 and 25; 21 and 28. In some embodiments,a first macrocycle-forming linker connects amino acid pairs 4 and 8; 5and 12; 8 and 12; 8 and 15; 9 and 16; 12 and 16; or 12 and 19; and asecond macrocycle-forming linker connects amino acid pairs 15 and 22; 18and 25; 21 and 25; 21 and 28; 22 and 29; or 25 and 29. For example, thefirst macrocycle-forming linker connects amino acid pairs 4 and 8; 5 and12; or 12 and 19; and the second macrocycle-forming linker connectsamino acid pairs 15 and 22; 18 and 25; 21 and 25; or 21 and 28. In someembodiments, the first macrocycle-forming linker connects amino acidpairs 4 and 8 and the second macrocycle-forming linker connects aminoacid pairs 21 and 25.

In some embodiments, a peptidomimetic macrocycle comprises an amino acidsequence which is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to GHRH 1-44, GHRH 1-29 and/or to anamino acid sequence chosen from the group consisting of the amino acidsequences in Table 1, 2 or 4, and further comprises a macrocycle-forminglinker connecting a first amino acid to a second amino acid, wherein thefirst and second amino acids are selected from the following pairs ofamino acids: 4 and 8; 5 and 12; 8 and 12; 8 and 15; 9 and 16; 12 and 16;12 and 19; 15 and 22; 18 and 25; 21 and 25; 21 and 28; 22 and 29. Forexample, the macrocycle-forming linker connects amino acids 12 and 19.

In some embodiments, a peptidomimetic macrocycle comprises a sequencechosen from the group consisting of the amino acid sequences in Tables1, 2 or 4, or the amino acid sequence of the peptidomimetic macrocycleis chosen from the group consisting of the amino acid sequences inTables 1, 2 or 4.

In some embodiments, the peptidomimetic macrocycle comprises a helix,such as an α-helix or a 3₁₀ helix. In other embodiments, thepeptidomimetic macrocycle comprises an α,α-disubstituted amino acid. Forexample, at least one amino acid, or each amino acid, connected by themacrocycle-forming linker is an α,α-disubstituted amino acid.

In some embodiments, a peptidomimetic macrocycle of the inventioncomprises a crosslinker linking the α-positions of at least two aminoacids.

In some embodiments, the peptidomimetic macrocycle has the formula:

wherein:

each A, C, D, and E is independently an amino acid;

B is an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

and wherein A, B, C, D, and E, taken together with the crosslinked aminoacids connected by the macrocycle-forming linker L, form the amino acidsequence of the peptidomimetic macrocycle which is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toGHRH 1-44, GHRH 1-29 and/or to an amino acid sequence chosen from thegroup consisting of the amino acid sequences in Table 1, 2 or 4;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v and w are independently integers from 1-1000, for example 1-500,1-200, 1-100, 1-50, 1-40, 1-25, 1-20, 1 to 15, or 1 to 10;

u, x, y and z are independently integers from 0-10, for example u is 1,2, or 3; and

n is an integer from 1-5. For example, u is 2. In some embodiments, thesum of x+y+z is 2, 3 or 6, for example 3 or 6.

In some embodiments, the peptidomimetic macrocycle of Formula (I) hasthe Formula:

wherein each A, C, D, and E is independently an amino acid;

B is an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

L′ is a macrocycle-forming linker of the formula -L₁′-L₂′-;

and wherein A, B, C, D, and E, taken together with the crosslinked aminoacids connected by the macrocycle-forming linkers L and L′, form theamino acid sequence of the peptidomimetic macrocycle;

R₁′ and R₂′ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

L₁′ and L₂′ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;

R₇′ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈′ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v′ and w′ are independently integers from 1-1000, for example 1-500,1-200, 1-100, 1-50, 1-40, 1-25, 1-20, 1 to 15, or 1 to 10;

x′, y′ and z′ are independently integers from 0-10; and

n is an integer from 1-5. In some embodiments, the sum of x′+y′+z′ is 2,3 or 6, for example 3 or 6.

In some embodiments of any of the peptidomimetic macrocycles describedherein, each K is O, S, SO, SO₂, CO, or CO₂.

In other embodiments, the peptidomimetic macrocycle may comprise acrosslinker linking a backbone amino group of a first amino acid to asecond amino acid within the peptidomimetic macrocycle. For example, theinvention provides peptidomimetic macrocycles of the Formula (II) or(IIa):

wherein:

each A, C, D, and E is independently an amino acid;

B is an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

and wherein A, B, C, D, and E, taken together with the crosslinked aminoacids connected by the macrocycle-forming linker -L₁-L₂-, form the aminoacid sequence of the peptidomimetic macrocycle which is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identicalto GHRH 1-44, GHRH 1-29 and/or to an amino acid sequence chosen from thegroup consisting of the amino acid sequences in Table 1, 2 or 4;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

v and w are independently integers from 1-1000, for example 1-500,1-200, 1-100, 1-50, 1-40, 1-25, 1-20, 1 to 15, or 1 to 10;

u, x, y and z are independently integers from 0-10, for example u is1-3; and

n is an integer from 1-5.

Also provided herein is a peptidomimetic macrocycle comprising an aminoacid sequence of Formula:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29  (SEQID NO: 1)

wherein:

X1 is Tyr or His;

X2 is Ala, D-Ala, or Val;

X3 is Asp;

X4 is Ala or a crosslinked amino acid;

X5 is Ile;

X6 is Phe;

X7 is Thr;

X8 is Gln, Asn, or a crosslinked amino acid;

X9 is Ser or a crosslinked amino acid;

X10 is Tyr;

X11 is Arg, Ala or Gln;

X12 is Lys, Ala, Gln or a crosslinked amino acid;

X13 is Val or lie;

X14 is Leu;

X15 is Gly, Ala or a crosslinked amino acid;

X16 is Gln, Glu or a crosslinked amino acid;

X17 is Leu;

X18 is Ser, Tyr or a crosslinked amino acid;

X19 is Ala or a crosslinked amino acid;

X20 is Arg or Gln;

X21 is Lys, Gln or a crosslinked amino acid;

X22 is Leu, Ala, or a crosslinked amino acid;

X23 is Leu;

X24 is Gln, Glu or His;

X25 is Asp, Glu or a crosslinked amino acid;

X26 is Ile;

X27 is Met, Ile, Leu or Nle;

X28 is Ser or a crosslinked amino acid;

X29 is Arg, Ala, Gln or a crosslinked amino acid;

wherein the peptidomimetic macrocycle comprises at least onemacrocycle-forming linker connecting at least one pair of amino acidsselected from X1-X29;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, or CO₂;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent; and

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent.

For example, the polypeptide comprises at least one, or at least two,macrocycle-forming linkers which connect one of the following pairs ofamino acids: X4 and X8; X5 and X12; X8 and X12; X8 and X15; X9 and X16;X12 and X16; X12 and X19; X15 and X22; X18 and X25; X21 and X25; X21 andX28; X22 and X29; X25 and X29. For example, each macrocycle-forminglinker connects one of the following pairs of amino acids: X4 and X8; X5and X12; X12 and X19; X15 and X22; X18 and X25; X21 and X25; X21 andX28.

In some embodiments, peptidomimetic macrocycles comprise amacrocycle-forming linker of Formula -L₁-L₂-, wherein L₁ and L₂ areindependently alkylene, alkenylene or alkynylene. For example, L₁ and L₂are independently C₃-C₁₀ alkylene or alkenylene, or C₃-C₆ alkylene oralkenylene.

In some embodiments, R₁ and R₂ are independently H or alkyl, for examplemethyl.

Additionally, the invention provides a method of increasing thecirculating level of growth hormone (GH) in a subject, a method ofincreasing lean muscle mass in a subject, and a method of reducingadipose tissue (such as abdominal adipose tissue) in a subjectcomprising administering to the subject a peptidomimetic macrocycle ofthe invention. For example, subjects suffering from obesity, includingabdominal obesity, are treated using a peptidomimetic macrocycle of theinvention. The invention also provides a method of treating musclewasting diseases that include anorexias, cachexias (such as cancercachexia, chronic heart failure cachexia, chronic obstructive pulmonarydisease cachexia, rheumatoid arthritis cachexia) and sarcopenias, amethod of treating lipodystrophies that include HIV lipodystrophy, amethod of treating growth hormone disorders that include adult andpediatric growth hormone deficiencies, or a method of treatinggastroparesis or short bowel syndrome. Pediatric growth hormonedeficiency may be, for example, linked with or associated to idiopathicshort stature, SGA (infant small for gestational age), chronic kidneydisease, Prader-Willi syndrome Turner syndrome, short stature homeobox(SHOX) gene deficiency, or primary IGF-1 deficiency. The invention alsoprovides a method of treating muscle wasting diseases, lipodystrophies,growth hormone disorders or gastroparesis/short bowel syndrome in asubject by administering an agonist of the GHRH receptor, such as ananalog of GHRH, wherein the agonist is administered no more frequentlythan once daily, no more frequently than every other day, no morefrequently than twice weekly, no more frequently than weekly, or no morefrequently than every other week. The invention also provides a methodof increasing the circulating level of growth hormone (GH) in a subjectby administering an agonist of the GHRH receptor, such as an analog ofGHRH, wherein the agonist is administered no more frequently than oncedaily, no more frequently than every other day, no more frequently thantwice weekly, no more frequently than weekly, or no more frequently thanevery other week.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A and 1B show improved stabilities to trypsin proteolysis of thepeptidomimetic macrocycles of the invention.

FIG. 2 shows improved serum stabilities of the peptidomimeticmacrocycles of the invention.

FIGS. 3 and 3 a show GHRH receptor agonist activities measured by cAMPrelease and trypsin half-lives of the peptidomimetic macrocycles of theinvention. For cAMP values, “+” represents values greater than 50 nM;“++” represents values between 10-50 nM; “+++” represents values between1-10 nM; “++++” represents values lower than 1 nM. For trypsinhalf-lives, “+” represents values lower than 50 min.; “++” representsvalues between 50-100 min.; “+++” represents values between 100-200min.; “++++” represents values greater than 200 min.; and “NT” signifies“not tested”. FIG. 3 discloses SEQ ID NOS 89-131, respectively, in orderof appearance. FIG. 3a discloses SEQ ID NOS 132-137, respectively, inorder of appearance.

FIG. 4 shows the result of a plasma PK study performed withpeptidomimetic macrocycle SP-1.

FIG. 5 shows the result of a plasma PK study performed withpeptidomimetic macrocycle SP-8.

FIG. 6 shows the result of a plasma PK study performed withpeptidomimetic macrocycle SP-6.

FIG. 7 shows the result of a plasma PK study performed withpeptidomimetic macrocycle SP-21.

FIG. 8 shows the result of a plasma PK study performed withpeptidomimetic macrocycle SP-32.

FIG. 9 shows the result of a plasma PK study performed withpeptidomimetic macrocycles SP-1, SP-6, SP-8, SP-21, and SP-32.

FIG. 10 shows stimulation of growth hormone production by peptidomimeticmacrocycle SP-8.

FIG. 11 shows growth hormone release (AUC) induced by sermorelin incomparison to peptidomimetic macrocycles SP-1, SP-6, SP-8, SP-21, andSP-32.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “macrocycle” refers to a molecule having achemical structure including a ring or cycle formed by at least 9covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” or “crosslinkedpolypeptide” refers to a compound comprising a plurality of amino acidresidues joined by a plurality of peptide bonds and at least onemacrocycle-forming linker which forms a macrocycle between a firstnaturally-occurring or non-naturally-occurring amino acid residue (oranalog) and a second naturally-occurring or non-naturally-occurringamino acid residue (or analog) within the same molecule. Peptidomimeticmacrocycle include embodiments where the macrocycle-forming linkerconnects the α carbon of the first amino acid residue (or analog) to theα carbon of the second amino acid residue (or analog). Thepeptidomimetic macrocycles optionally include one or more non-peptidebonds between one or more amino acid residues and/or amino acid analogresidues, and optionally include one or more non-naturally-occurringamino acid residues or amino acid analog residues in addition to anywhich form the macrocycle. A “corresponding uncrosslinked polypeptide”when referred to in the context of a peptidomimetic macrocycle isunderstood to relate to a polypeptide of the same length as themacrocycle and comprising the equivalent natural amino acids of thewild-type sequence corresponding to the macrocycle.

As used herein, the term “stability” refers to the maintenance of adefined secondary structure in solution by a peptidomimetic macrocycleof the invention as measured by circular dichroism, NMR or anotherbiophysical measure, or resistance to proteolytic degradation in vitroor in vivo. Non-limiting examples of secondary structures contemplatedin this invention are α-helices, 3₁₀ helices, β-turns, and β-pleatedsheets.

As used herein, the term “helical stability” refers to the maintenanceof a helical structure by a peptidomimetic macrocycle of the inventionas measured by circular dichroism or NMR. For example, in someembodiments, the peptidomimetic macrocycles of the invention exhibit atleast a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as determinedby circular dichroism compared to a corresponding uncrosslinkedmacrocycle.

The term “amino acid” refers to a molecule containing both an aminogroup and a carboxyl group. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. The term amino acid, asused herein, includes without limitation, α-amino acids, natural aminoacids, non-natural amino acids, and amino acid analogs.

The term “α-amino acid” refers to a molecule containing both an aminogroup and a carboxyl group bound to a carbon which is designated theα-carbon.

The term “β-amino acid” refers to a molecule containing both an aminogroup and a carboxyl group in a β configuration.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The following table shows a summary of the properties of natural aminoacids:

3- 1- Side-chain Letter Letter Side-chain charge Hydropathy Amino AcidCode Code Polarity (pH 7.4) Index Alanine Ala A nonpolar neutral 1.8Arginine Arg R polar positive −4.5 Asparagine Asn N polar neutral −3.5Aspartic acid Asp D polar negative −3.5 Cysteine Cys C polar neutral 2.5Glutamic acid Glu E polar negative −3.5 Glutamine Gln Q polar neutral−3.5 Glycine Gly G nonpolar neutral −0.4 Histidine His H polar positive(10%) −3.2 neutral (90%) Isoleucine Ile I nonpolar neutral 4.5 LeucineLeu L nonpolar neutral 3.8 Lysine Lys K polar positive −3.9 MethionineMet M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8Proline Pro P nonpolar neutral −1.6 Serine Ser S polar neutral −0.8Threonine Thr T polar neutral −0.7 Tryptophan Trp W nonpolar neutral−0.9 Tyrosine Tyr Y polar neutral −1.3 Valine Val V nonpolar neutral 4.2

“Hydrophobic amino acids” include small hydrophobic amino acids andlarge hydrophobic amino acids. “Small hydrophobic amino acid” areglycine, alanine, proline, and analogs thereof “Large hydrophobic aminoacids” are valine, leucine, isoleucine, phenylalanine, methionine,tryptophan, and analogs thereof. “Polar amino acids” are serine,threonine, asparagine, glutamine, cysteine, tyrosine, and analogsthereof. “Charged amino acids” are lysine, arginine, histidine,aspartate, glutamate, and analogs thereof.

The term “amino acid analog” refers to a molecule which is structurallysimilar to an amino acid and which can be substituted for an amino acidin the formation of a peptidomimetic macrocycle. Amino acid analogsinclude, without limitation, 3-amino acids and amino acids where theamino or carboxy group is substituted by a similarly reactive group(e.g., substitution of the primary amine with a secondary or tertiaryamine, or substitution of the carboxy group with an ester).

The term “non-natural amino acid” refers to an amino acid which is notone of the twenty amino acids commonly found in peptides synthesized innature, and known by the one letter abbreviations A, R, N, C, D, Q, E,G, H, I, L, K, M, F, P, S, T, W, Y and V. Non-natural amino acids oramino acid analogs include, without limitation, structures according tothe following:

Amino acid analogs include β-amino acid analogs. Examples of β-aminoacid analogs include, but are not limited to, the following: cyclicβ-amino acid analogs; β-alanine; (R)-β-phenylalanine;(R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;(R)-3-amino-4-(1-naphthyl)-butyric acid;(R)-3-amino-4-(2,4-dichlorophenyl)butyric acid;(R)-3-amino-4-(2-chlorophenyl)-butyric acid;(R)-3-amino-4-(2-cyanophenyl)-butyric acid;(R)-3-amino-4-(2-fluorophenyl)-butyric acid;(R)-3-amino-4-(2-furyl)-butyric acid;(R)-3-amino-4-(2-methylphenyl)-butyric acid;(R)-3-amino-4-(2-naphthyl)-butyric acid;(R)-3-amino-4-(2-thienyl)-butyric acid;(R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-(3,4-dichlorophenyl)butyric acid;(R)-3-amino-4-(3,4-difluorophenyl)butyric acid;(R)-3-amino-4-(3-benzothienyl)-butyric acid;(R)-3-amino-4-(3-chlorophenyl)-butyric acid;(R)-3-amino-4-(3-cyanophenyl)-butyric acid;(R)-3-amino-4-(3-fluorophenyl)-butyric acid;(R)-3-amino-4-(3-methylphenyl)-butyric acid;(R)-3-amino-4-(3-pyridyl)-butyric acid;(R)-3-amino-4-(3-thienyl)-butyric acid;(R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-(4-bromophenyl)-butyric acid;(R)-3-amino-4-(4-chlorophenyl)-butyric acid;(R)-3-amino-4-(4-cyanophenyl)-butyric acid;(R)-3-amino-4-(4-fluorophenyl)-butyric acid;(R)-3-amino-4-(4-iodophenyl)-butyric acid;(R)-3-amino-4-(4-methylphenyl)-butyric acid;(R)-3-amino-4-(4-nitrophenyl)-butyric acid;(R)-3-amino-4-(4-pyridyl)-butyric acid;(R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-pentafluoro-phenylbutyric acid; (R)-3-amino-5-hexenoicacid; (R)-3-amino-5-hexynoic acid; (R)-3-amino-5-phenylpentanoic acid;(R)-3-amino-6-phenyl-5-hexenoic acid;(S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;(S)-3-amino-4-(1-naphthyl)-butyric acid;(S)-3-amino-4-(2,4-dichlorophenyl)butyric acid;(S)-3-amino-4-(2-chlorophenyl)-butyric acid;(S)-3-amino-4-(2-cyanophenyl)-butyric acid;(S)-3-amino-4-(2-fluorophenyl)-butyric acid;(S)-3-amino-4-(2-furyl)-butyric acid;(S)-3-amino-4-(2-methylphenyl)-butyric acid;(S)-3-amino-4-(2-naphthyl)-butyric acid;(S)-3-amino-4-(2-thienyl)-butyric acid;(S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;(S)-3-amino-4-(3,4-dichlorophenyl)butyric acid;(S)-3-amino-4-(3,4-difluorophenyl)butyric acid;(S)-3-amino-4-(3-benzothienyl)-butyric acid;(S)-3-amino-4-(3-chlorophenyl)-butyric acid;(S)-3-amino-4-(3-cyanophenyl)-butyric acid;(S)-3-amino-4-(3-fluorophenyl)-butyric acid;(S)-3-amino-4-(3-methylphenyl)-butyric acid;(S)-3-amino-4-(3-pyridyl)-butyric acid;(S)-3-amino-4-(3-thienyl)-butyric acid;(S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;(S)-3-amino-4-(4-bromophenyl)-butyric acid;(S)-3-amino-4-(4-chlorophenyl)-butyric acid;(S)-3-amino-4-(4-cyanophenyl)-butyric acid;(S)-3-amino-4-(4-fluorophenyl)-butyric acid;(S)-3-amino-4-(4-iodophenyl)-butyric acid;(S)-3-amino-4-(4-methylphenyl)-butyric acid;(S)-3-amino-4-(4-nitrophenyl)-butyric acid;(S)-3-amino-4-(4-pyridyl)-butyric acid;(S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;(S)-3-amino-4-pentafluoro-phenylbutyric acid; (S)-3-amino-5-hexenoicacid; (S)-3-amino-5-hexynoic acid; (S)-3-amino-5-phenylpentanoic acid;(S)-3-amino-6-phenyl-5-hexenoic acid;1,2,5,6-tetrahydropyridine-3-carboxylic acid;1,2,5,6-tetrahydropyridine-4-carboxylic acid;3-amino-3-(2-chlorophenyl)-propionic acid;3-amino-3-(2-thienyl)-propionic acid;3-amino-3-(3-bromophenyl)-propionic acid;3-amino-3-(4-chlorophenyl)-propionic acid;3-amino-3-(4-methoxyphenyl)-propionic acid;3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid;D-β-phenylalanine; β-leucine; L-β-homoalanine; L-β-homoaspartic acidγ-benzyl ester; L-β-homoglutamic acid δ-benzyl ester;L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionine;L-β-homophenylalanine; L-β-homoproline; L-β-homotryptophan;L-β-homovaline; L-Nω-benzyloxycarbonyl-β-homolysine;Nω-L-β-homoarginine; O-benzyl-L-β-homohydroxyproline;O-benzyl-L-β-homoserine; O-benzyl-L-β-homothreonine;O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoasparagine;(R)-β-phenylalanine; L-β-homoaspartic acid γ-t-butyl ester;L-β-homoglutamic acid δ-t-butyl ester; L-Nω-β-homolysine;Nδ-trityl-L-β-homoglutamine;Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine;O-t-butyl-L-β-homohydroxy-proline; O-t-butyl-L-β-homoserine;O-t-butyl-L-β-homothreonine; O-t-butyl-L-β-homotyrosine;2-aminocyclopentane carboxylic acid; and 2-aminocyclohexane carboxylicacid.

Amino acid analogs include analogs of alanine, valine, glycine orleucine. Examples of amino acid analogs of alanine, valine, glycine, andleucine include, but are not limited to, the following:α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid;α-methyl-leucine; β-(1-naphthyl)-D-alanine; β-(1-naphthyl)-L-alanine;β-(2-naphthyl)-D-alanine; β-(2-naphthyl)-L-alanine;1-(2-pyridyl)-D-alanine; β-(2-pyridyl)-L-alanine;β-(2-thienyl)-D-alanine; β-(2-thienyl)-L-alanine;β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine;β-(3-pyridyl)-D-alanine; β-(3-pyridyl)-L-alanine;β-(4-pyridyl)-D-alanine; β-(4-pyridyl)-L-alanine; 1-chloro-L-alanine;1-cyano-L-alanin; 3-cyclohexyl-D-alanine; 3-cyclohexyl-L-alanine;3-cyclopenten-1-yl-alanine; 3-cyclopentyl-alanine;3-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt; β-t-butyl-D-alanine;β-t-butyl-L-alanine; γ-aminobutyric acid; L-α,β-diaminopropionic acid;2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine;2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine;3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine;4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium salt;4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine;4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoicacid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt;cyclopentyl-Gly-OH.dicyclohexylammonium salt; D-α,β-diaminopropionicacid; D-α-aminobutyric acid; D-α-t-butylglycine; D-(2-thienyl)glycine;D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine;D-allylglycine.dicyclohexylammonium salt; D-cyclohexylglycine;D-norvaline; D-phenylglycine; β-aminobutyric acid; β-aminoisobutyricacid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine;(2-methylphenyl)glycine; (2-thiazoyl)glycine; (2-thienyl)glycine;2-amino-β-(dimethylamino)-propionic acid; L-α,β-diaminopropionic acid;L-α-aminobutyric acid; L-α-t-butylglycine; L-β-thienyl)glycine;L-2-amino-β-(dimethylamino)-propionic acid; L-2-aminocaproic aciddicyclohexyl-ammonium salt; L-2-indanylglycine;L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine;L-phenylglycine; L-propargylglycine; L-norvaline;N-α-aminomethyl-L-alanine; D-α,γ-diaminobutyric acid;L-α,γ-diaminobutyric acid; β-cyclopropyl-L-alanine;(N-β-(2,4-dinitrophenyl))-L-α,β-diaminopropionic acid;(N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,β-diaminopropionicacid;(N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,β-diaminopropionicacid; (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid;(N-β-allyloxycarbonyl)-L-α,β-diaminopropionic acid;(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyricacid;(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyricacid; (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid;(N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid;(N-γ-allyloxycarbonyl)-L-α,γ-diaminobutyric acid; D-α,γ-diaminobutyricacid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH;D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine;L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; andN-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.

Amino acid analogs include analogs of arginine or lysine. Examples ofamino acid analogs of arginine and lysine include, but are not limitedto, the following: citrulline; L-2-amino-3-guanidinopropionic acid;L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me)₂-OH; Lys(N₃)—OH;Nδ-benzyloxycarbonyl-L-omithine; Nω-nitro-D-arginine;Nω-nitro-L-arginine; α-methyl-omithine; 2,6-diaminoheptanedioic acid;L-omithine;(Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-omithine;(Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-omithine;(Nδ-4-methyltrityl)-D-omithine; (Nδ-4-methyltrityl)-L-omithine;D-omithine; L-omithine; Arg(Me)(Pbf)-OH; Arg(Me)₂-OH (asymmetrical);Arg(Me)2-OH (symmetrical); Lys(ivDde)-OH; Lys(Me)2-OH.HCl; Lys(Me3)-OHchloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

Amino acid analogs include analogs of aspartic or glutamic acids.Examples of amino acid analogs of aspartic and glutamic acids include,but are not limited to, the following: α-methyl-D-aspartic acid;α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamicacid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid;2,6-diaminopimelic acid; L-α-aminosuberic acid; D-2-aminoadipic acid;D-α-aminosuberic acid; α-aminopimelic acid; iminodiacetic acid;L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamicacid γ,γ-di-t-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-t-butylester; Glu(OAll)-OH; L-Asu(OtBu)—OH; and pyroglutamic acid.

Amino acid analogs include analogs of cysteine and methionine. Examplesof amino acid analogs of cysteine and methionine include, but are notlimited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, α-methyl-methionine,Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH,2-amino-4-(ethylthio)butyric acid, buthionine, buthioninesulfoximine,ethionine, methionine methylsulfonium chloride, selenomethionine,cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine,[2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine,4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine,4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine,benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine,carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine,methyl-L-cysteine, t-butyl-D-cysteine, trityl-L-homocysteine,trityl-D-penicillamine, cystathionine, homocystine, L-homocystine,(2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine,Cys(StBu)—OH, and acetamidomethyl-D-penicillamine.

Amino acid analogs include analogs of phenylalanine and tyrosine.Examples of amino acid analogs of phenylalanine and tyrosine include3-methyl-phenylalanine, 3-hydroxyphenylalanine,α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine,α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine,2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine,2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine,2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine,2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine,2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine,2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine,2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine,3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine,3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine,3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine,3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine,3,5,3′-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine,3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine,3-(trifluoromethyl)-D-phenylalanine,3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine,3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine,3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine,3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine,3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine,3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine,3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine,3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine,3-nitro-L-phenylalanine, 3-nitro-L-tyrosine,4-(trifluoromethyl)-D-phenylalanine,4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine,4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine,4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine,4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine,4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine,4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine,4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine,4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine,thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tyrosine, andmethyl-tyrosine.

Amino acid analogs include analogs of proline. Examples of amino acidanalogs of proline include, but are not limited to, 3,4-dehydro-proline,4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid,and trans-4-fluoro-proline.

Amino acid analogs include analogs of serine and threonine. Examples ofamino acid analogs of serine and threonine include, but are not limitedto, 3-amino-2-hydroxy-5-methylhexanoic acid,2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid,2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoicacid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionicacid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid,and α-methylserine.

Amino acid analogs include analogs of tryptophan. Examples of amino acidanalogs of tryptophan include, but are not limited to, the following:α-methyl-tryptophan; β-(3-benzothienyl)-D-alanine;β-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan;5-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan;5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan;5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan;6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan;6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan;7-bromo-tryptophan; 7-methyl-tryptophan;D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

In some embodiments, amino acid analogs are racemic. In someembodiments, the D isomer of the amino acid analog is used. In someembodiments, the L isomer of the amino acid analog is used. In otherembodiments, the amino acid analog comprises chiral centers that are inthe R or S configuration. In still other embodiments, the amino group(s)of a β-amino acid analog is substituted with a protecting group, e.g.,tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC),tosyl, and the like. In yet other embodiments, the carboxylic acidfunctional group of a β-amino acid analog is protected, e.g., as itsester derivative. In some embodiments the salt of the amino acid analogis used.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide without abolishing orsubstantially abolishing its essential biological or biochemicalactivity (e.g., receptor binding or activation). An “essential” aminoacid residue is a residue that, when altered from the wild-type sequenceof the polypeptide, results in abolishing or substantially abolishingthe polypeptide's essential biological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a polypeptide, for example, isreplaced with another amino acid residue from the same side chainfamily. Other examples of acceptable substitutions are substitutionsbased on isosteric considerations (e.g. norleucine for methionine) orother properties (e.g. 2-thienylalanine for phenylalanine).

The term “capping group” refers to the chemical moiety occurring ateither the carboxy or amino terminus of the polypeptide chain of thesubject peptidomimetic macrocycle. The capping group of a carboxyterminus includes an unmodified carboxylic acid (ie —COOH) or acarboxylic acid with a substituent. For example, the carboxy terminuscan be substituted with an amino group to yield a carboxamide at theC-terminus. Various substituents include but are not limited to primaryand secondary amines, including pegylated secondary amines.Representative secondary amine capping groups for the C-terminusinclude:

The capping group of an amino terminus includes an unmodified amine (ie—NH₂) or an amine with a substituent. For example, the amino terminuscan be substituted with an acyl group to yield a carboxamide at theN-terminus. Various substituents include but are not limited tosubstituted acyl groups, including C₁-C₆ carbonyls, C₇-C₃₀ carbonyls,and pegylated carbamates. Representative capping groups for theN-terminus include:

The term “member” as used herein in conjunction with macrocycles ormacrocycle-forming linkers refers to the atoms that form or can form themacrocycle, and excludes substituent or side chain atoms. By analogy,cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are allconsidered ten-membered macrocycles as the hydrogen or fluorosubstituents or methyl side chains do not participate in forming themacrocycle.

The symbol “

” when used as part of a molecular structure refers to a single bond ora trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon (or another backbone atom) in an amino acid. For example, theamino acid side chain for alanine is methyl, the amino acid side chainfor phenylalanine is phenylmethyl, the amino acid side chain forcysteine is thiomethyl, the amino acid side chain for aspartate iscarboxymethyl, the amino acid side chain for tyrosine is4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acidside chains are also included, for example, those that occur in nature(e.g., an amino acid metabolite) or those that are made synthetically(e.g., an α,α di-substituted amino acid).

The term “α,α di-substituted amino” acid refers to a molecule or moietycontaining both an amino group and a carboxyl group bound to a carbon(the α-carbon) that is attached to two natural or non-natural amino acidside chains.

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full lengthproteins (e.g., fully processed proteins) as well as shorter amino acidsequences (e.g., fragments of naturally-occurring proteins or syntheticpolypeptide fragments).

The term “macrocyclization reagent” or “macrocycle-forming reagent” asused herein refers to any reagent which may be used to prepare apeptidomimetic macrocycle of the invention by mediating the reactionbetween two reactive groups. Reactive groups may be, for example, anazide and alkyne, in which case macrocyclization reagents include,without limitation, Cu reagents such as reagents which provide areactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II)salts such as Cu(CO₂CH₃)₂, CuSO₄, and CuCl₂ that can be converted insitu to an active Cu(I) reagent by the addition of a reducing agent suchas ascorbic acid or sodium ascorbate. Macrocyclization reagents mayadditionally include, for example, Ru reagents known in the art such asCp*RuCl(PPh₃)₂, [Cp*RuCl]₄ or other Ru reagents which may provide areactive Ru(II) species. In other cases, the reactive groups areterminal olefins. In such embodiments, the macrocyclization reagents ormacrocycle-forming reagents are metathesis catalysts including, but notlimited to, stabilized, late transition metal carbene complex catalystssuch as Group VIII transition metal carbene catalysts. For example, suchcatalysts are Ru and Os metal centers having a +2 oxidation state, anelectron count of 16 and pentacoordinated. In other examples, catalystshave W or Mo centers. Various catalysts are disclosed in Grubbs et al.,“Ring Closing Metathesis and Related Processes in Organic Synthesis”Acc. Chem. Res. 1995, 28, 446-452, and U.S. Pat. Nos. 5,811,515;7,932,397; U.S. Application No. 2011/0065915; U.S. Application No.2011/0245477; Yu et al., “Synthesis of Macrocyclic Natural Products byCatalyst-Controlled Stereoselective Ring-Closing Metathesis,” Nature2011, 479, 88; and Peryshkov et al., “Z-Selective Olefin MetathesisReactions Promoted by Tungsten Oxo Alkylidene Complexes,” J. Am. Chem.Soc. 2011, 133, 20754. In yet other cases, the reactive groups are thiolgroups. In such embodiments, the macrocyclization reagent is, forexample, a linker functionalized with two thiol-reactive groups such ashalogen groups.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine oriodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring aresubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkoxy” refers to an alkoxysubstituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkylgroup, as defined above. Representative examples of an arylalkyl groupinclude, but are not limited to, 2-methylphenyl, 3-methylphenyl,4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl,2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl,3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl,4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH2-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl,

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above,wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replacedwith a heterocycle. Representative examples of an alkylheterocycle groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—C(O)NH₂ group. Representative examples of an alkylamido group include,but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂,—CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂,—CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃,—C(CH₃)₂CH₂C(O)NH₂, —CH₂—CH₂—NH—C(O)—CH₃, —CH₂—CH₂—NH—C(O)—CH₃—CH3, and—CH₂—CH₂—NH—C(O)—CH═CH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein oneof the C₁-C₅ alkyl group's hydrogen atoms has been replaced with ahydroxyl group. Representative examples of an alkanol group include, butare not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—COOH group. Representative examples of an alkylcarboxy group include,but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Somecycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom orgroup such as a hydrogen atom on any molecule, compound or moiety.Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds of this invention contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are included in thepresent invention unless expressly provided otherwise. In someembodiments, the compounds of this invention are also represented inmultiple tautomeric forms, in such instances, the invention includes alltautomeric forms of the compounds described herein (e.g., if alkylationof a ring system results in alkylation at multiple sites, the inventionincludes all such reaction products). All such isomeric forms of suchcompounds are included in the present invention unless expresslyprovided otherwise. All crystal forms of the compounds described hereinare included in the present invention unless expressly providedotherwise.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.1) increase ordecrease of at least 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the variable is equal to any of the valueswithin that range. Thus, for a variable which is inherently discrete,the variable is equal to any integer value within the numerical range,including the end-points of the range. Similarly, for a variable whichis inherently continuous, the variable is equal to any real value withinthe numerical range, including the end-points of the range. As anexample, and without limitation, a variable which is described as havingvalues between 0 and 2 takes the values 0, 1 or 2 if the variable isinherently discrete, and takes the values 0.0, 0.1, 0.01, 0.001, or anyother real values ≥0 and ≤2 if the variable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The term “on average” represents the mean value derived from performingat least three independent replicates for each data point.

The term “biological activity” encompasses structural and functionalproperties of a macrocycle of the invention. Biological activity is, forexample, structural stability, alpha-helicity, affinity for a target,resistance to proteolytic degradation, cell penetrability, intracellularstability, in vivo stability, or any combination thereof.

The details of one or more particular embodiments of the invention areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

In some embodiments, the peptide sequences are derived from a GHRHpeptide. For example, the peptide sequences are derived from human GHRH(1-29) or human GHRH (1-44).

A non-limiting exemplary list of suitable GHRH peptides for use in thepresent invention is given in Table 1 and Table 2 below. In Tables 1 and2, all peptides possess a free amino terminus (shown as H—) and allpeptides possess an carboxamide terminus (shown as —NH2). X residuesform cross-links to one other X residue, Z residues form cross-links toone other Z residue, and XX residues form cross-links with two other Xresidues. In Tables 1 and 2, amino acid A2 is either L-Ala or D-Ala, A8is either L-Asn or L-Gln, A15 is either L-Ala or Gly, and A27 is eitherL-Nle or L-Leu.

TABLE 1 (SEQ ID NOS 2-74, respectively, in order of appearance)H-Y-A2-D-X-IFT-X-SYRKVL-A15-QLSAR-Z-LLQ-Z-I-A27- SR-NH2H-Y-A2-D-X-IFT-X-SYRKVL-A15-QLSARKLLQ-Z-I-A27-S- Z-NH2H-Y-A2-DAIFT-X-SYR-X-VL-A15-QLSAR-Z-LLQ-Z-I-A27- SR-NH2H-Y-A2-DAIFT-X-SYR-X-VL-A15-QLSARKLLQ-Z-I-A27-S- Z-NH2H-Y-A2-DAIFT-A8-SY-X-KVL-X-QLSAR-Z-LLQ-Z-I-A27- SR-NH2H-Y-A2-DAIFT-A8-SY-X-KVL-X-QLSARKLLQ-Z-I-A27-S- Z-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-X-LSAR-Z-LLQ-Z-I- A27-SR-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-X-LSARKLLQ-Z-I-A27- S-Z-NH2H-Y-A2-D-X-IFT-A8-SY-X-KVL-A15-QLSARKLLQDI-A27- SR-NH2H-Y-A2-DA-X-FT-A8-SYR-X-VL-A15-QLSARKLLQDI-A27- SR-NH2H-Y-A2-DAI-X-T-A8-SYRK-X-L-A15-QLSARKLLQDI-A27- SR-NH2H-Y-A2-DAIF-X-A8-SYRKV-X-A15-QLSARKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-X-SYRKVL-X-QLSARKLLQDI-A27-SR-NH2H-Y-A2-DAIFT-A8-X-YRKVL-A15-X-LSARKLLQDI-A27-SR- NH2H-Y-A2-DAIFT-A8-S-X-RKVL-A15-Q-X-SARKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SY-X-KVL-A15-QL-X-ARKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-QLS-X-RKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRK-X-L-A15-QLSA-X-KLLQDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRKV-X-A15-QLSAR-X-LLQDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRKVL-X-QLSARK-X-LQDI-A27-SR- NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-X-LSARKL-X-QDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-Q-X-SARKLL-X-DI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-QL-X-ARKLLQ-X-I-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-QLS-X-RKLLQD-X-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-QLSA-X-KLLQDI-X-SR- NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-QLSAR-X-LLQDI-A27-X- R-NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-QLSARK-X-LQDI-A27-S- X-NH2H-Y-A2-D-X-IFT-A8-SY-X-KVL-A15-QLSAR-Z-LLQ-Z-I- A27-SR-NH2H-Y-A2-D-X-IFT-A8-SY-X-KVL-A15-QLSARKLLQ-Z-I- A27-S-Z-NH2H-Y-A2-DA-X-FT-A8-SYR-X-VL-A15-QLSAR-Z-LLQ-Z-I- A27-SR-NH2H-Y-A2-DA-X-FT-A8-SYR-X-VL-A15-QLSARKLLQ-Z-I- A27-S-Z-NH2H-Y-A2-DAI-X-T-A8-SYRK-X-L-A15-QLSAR-Z-LLQ-Z-I- A27-SR-NH2H-Y-A2-DAI-X-T-A8-SYRK-X-L-A15-QLSARKLLQ-Z-I-A27- S-Z-NH2H-Y-A2-DAIF-X-A8-SYRKV-X-A15-QLSAR-Z-LLQ-Z-I-A27- SR-NH2H-Y-A2-DAIF-X-A8-SYRKV-X-A15-QLSARKLLQ-Z-I-A27-S- Z-NH2H-Y-A2-DAIFT-X-SYRKVL-X-QLSAR-Z-LLQ-Z-I-A27-SR- NH2H-Y-A2-DAIFT-X-SYRKVL-X-QLSARKLLQ-Z-I-A27-S-Z- NH2H-Y-A2-DAIFT-A8-X-YRKVL-A15-X-LSARKLLQ-Z-I-A27- S-Z-NH2H-Y-A2-DAIFT-A8-X-YRKVL-A15-X-LSAR-Z-LLQ-Z-I-A27- SR-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-QLS-X-RKLLQ-Z-I-A27- S-Z-NH2H-Y-A2-D-X-IFT-X-SYRKVL-Z-QLSARK-Z-LQDI-A27-SR- NH2H-Y-A2-D-X-IFT-X-SYRKVL-A15-QL-Z-ARKLLQ-Z-I-A27- SR-NH2H-Y-A2-D-X-IFT-X-SYRKVL-A15-QLSAR-Z-LLQDI-A27-Z- R-NH2H-Y-A2-D-X-IFT-X-SYRKVL-A15-QLSARK-Z-LQDI-A27-S- Z-NH2H-Y-A2-DAIFT-X-SYR-X-VL-Z-QLSARK-Z-LQDI-A27-SR- NH2H-Y-A2-DAIFT-X-SYR-X-VL-A15-QL-Z-ARKLLQ-Z-I-A27- SR-NH2H-Y-A2-DAIFT-X-SYR-X-VL-A15-QLSAR-Z-LLQDI-A27-Z- R-NH2H-Y-A2-DAIFT-X-SYR-X-VL-A15-QLSARK-Z-LQDI-A27-S- Z-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-X-LSAR-Z-LLQDI-A27- Z-R-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-X-LSARK-Z-LQDI-A27- S-Z-NH2H-Y-A2-DAIFT-A8-SY-X-KVL-X-QLSAR-Z-LLQDI-A27-Z- R-NH2H-Y-A2-DAIFT-A8-SY-X-KVL-X-QLSARK-Z-LQDI-A27-S- Z-NH2H-Y-A2-D-X-IFT-XX-SYR-X-VL-A15-QLSARKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-X-SYR-XX-VL-A15-X-LSARKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-XX-LSA-X-KLLQDI- A27-SR-NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-QLSAR-X-LLQ-XX-I-A27- S-X-NH2H-Y-A2-D-X-IFT-XX-SYRKVL-X-QLSARKLLQDI-A27-SR- NH2H-Y-A2-D-X-IFT-A8-SY-XX-KVL-X-QLSARKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-X-SYR-XX-VL-A15-QLS-X-RKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-X-SYRKVL-XX-QLS-X-RKLLQDI-A27-SR- NH2H-Y-A2-DAIFT-A8-X-YRKVL-A15-XX-LSA-X-KLLQDI-A27- SR-NH2H-Y-A2-DAIFT-A8-X-YRK-XX-L-A15-QLSA-X-KLLQDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-QLS-XX-RKL-X-QDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-XX-LSARKL-X-QDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-X-LSARKL-XX-QDI-X-SR- NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-X-LSA-XX-KLLQDI-X-SR- NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-QL-X-ARK-XX-LQDI-A27-S- X-NH2H-Y-A2-DAIFT-A8-SYRKVL-A15-QL-X-ARKLLQ-XX-I-A27-S- X-NH2H-Y-A2-D-X-IFT-A8-SY-XX-KVL-A15-QL-X-ARKLLQDI-A27- SR-NH2H-Y-A2-DAIFT-X-SYRKVL-XX-QLSARK-X-LQDI-A27-SR-NH2H-Y-A2-DAIFT-A8-X-YRKVL-A15-XX-LSARKL-X-QDI-A27- SR-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15-QLS-XX-RKLLQD-X-A27- SR-NH2H-Y-A2-DAIFT-A8-SYRKVL-X-QLSARK-XX-LQDI-A27-S-X- NH2

TABLE 2 (SEQ ID NOS 75-88, respectively, in order of appearance)H-Y-A2-D-X-IFT-X-SYRKVL-A15-QLSAR-Z-LLQ-Z-I-A27- SRQQGESNQERGARARL-NH2H-Y-A2-D-X-IFT-X-SYRKVL-A15-QLSARKLLQ-Z-I-A27-S- Z-QQGESNQERGARARL-NH2H-Y-A2-D-X-IFT-X-SYRKVL-Z-QLSARK-Z-LQDI-A27- SRQQGESNQERGARARL-NH2H-Y-A2-D-X-IFT-X-SYRKVL-A15-QL-Z-ARKLLQ$I-A27- SRQQGESNQERGARARL-NH2H-Y-A2-D-X-IFT-X-SYRKVL-A15-QLSAR-Z-LLQDI-A27-- Z-RQQGESNQERGARARL-NH2H-Y-A2-D-X-IFT-X-SYRKVL-A15-QLSARK-Z-LQDI-A27- S-Z-QQGESNQERGARARL-NH2H-Y-A2-DAIFT-X-SYR-X-VL-A15-QLSAR-Z-LLQ-Z-I-A27- SRQQGESNQERGARARL-NH2H-Y-A2-DAIFT-X-SYR-X-VL-A15-QLSARKLLQ-Z-I-A27-S- Z-QQGESNQERGARARL-NH2H-Y-A2-DAIFT-A8-SY-X-KVL-X-QLSAR-Z-LLQ-Z-I-A27- SRQQGESNQERGARARL-NH2H-Y-A2-DAIFT-A8-SY-X-KVL-X-QLSARKLLQ-Z-I-A27-S- Z-QQGESNQERGARARL-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15--X-LSAR-Z-LLQ-Z-I-A27-SRQQGESNQERGARARL-NH2H-Y-A2-DAIFT-A8-SYR-X-VL-A15--X-LSARKLLQ-Z-I-A27-S-Z-QQGESNQERGARARL-NH2 H-Y-A2-DAIFT-X-SYRKVL-X-QLSAR-Z-LLQ-Z-I-A27-SRQQGESNQERGARARL-NH2 H-Y-A2-DAIFT-X-SYRKVL-X-QLSARKLLQ-Z-I-A27-S-Z-QQGESNQERGARARL-NH2

Peptidomimetic Macrocycles of the Invention

In some embodiments, a peptidomimetic macrocycle of the invention hasthe Formula (I):

wherein:

each A, C, D, and E is independently an amino acid (including natural ornon-natural amino acids and amino acid analogs) and the terminal D and Eindependently optionally include a capping group;

B is an amino acid (including natural or non-natural amino acids andamino acid analogs),

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-; and wherein A,B, C, D, and E, taken together with the crosslinked amino acidsconnected by the macrocycle-forming linker L, form the amino acidsequence of the peptidomimetic macrocycle which is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toGHRH 1-44, GHRH 1-29 and/or to an amino acid sequence chosen from thegroup consisting of the amino acid sequences in Table 1, 2 or 4;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]n, each being optionally substitutedwith R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v and w are independently integers from 1-1000, for example 1-500,1-200, 1-100, 1-50, 1-40, 1-25, 1-20, 1 to 15, or 1 to 10;

u, x, y and z are independently integers from 0-10, for example u is 1,2, or 3; and n is an integer from 1-5, for example 1. For example, u is2. In some embodiments, the sum of x+y+z is 2, 3 or 6, for example 3 or6.

In some embodiments, the peptidomimetic macrocycle of Formula (I) hasthe Formula:

wherein each A, C, D, and E is independently an amino acid;

B is an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

L′ is a macrocycle-forming linker of the formula -L₁′-L₂′-;

and wherein A, B, C, D, and E, taken together with the crosslinked aminoacids connected by the macrocycle-forming linkers L and L′, form theamino acid sequence of the peptidomimetic macrocycle;

R₁′ and R₂′ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

L₁′ and L₂′ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each K is independently O, S, SO, SO₂, CO, CO₂, or CONR₃;

R₇′ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈′ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v′ and w′ are independently integers from 1-1000, for example 1-500,1-200, 1-100, 1-50, 1-40, 1-25, 1-20, 1 to 15, or 1 to 10;

x′, y′ and z′ are independently integers from 0-10; and

n is an integer from 1-5. In some embodiments, the sum of x′+y′+z′ is 2,3 or 6, for example 3 or 6.

In some embodiments of any of the peptidomimetic macrocycles describedherein, each K is O, S, SO, SO₂, CO, or CO₂.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, the sum of the sum of x+y+z is atleast 3, and/or the sum of x′+y′+z′ is at least 3. In other embodimentsof the invention, the sum of the sum of x+y+z is 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 (for example 2, 3 or 6) and/or the sum of x′+y′+z′ is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 (for example 2, 3 or 6).

Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A]_(x), when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges. Similarly, when u is greater than 1, each compound of theinvention may encompass peptidomimetic macrocycles which are the same ordifferent. For example, a compound of the invention may comprisepeptidomimetic macrocycles comprising different linker lengths orchemical compositions.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Ca to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

In one embodiment, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl,arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the peptidomimetic macrocycle comprises astructure of Formula (I) which is:

In other embodiments, the peptidomimetic macrocycle of Formula (I) is acompound of any of the formulas shown below:

wherein “AA” represents any natural or non-natural amino acid side chainand “

” is [D]_(v), [E]_(w) as defined above, and n is an integer between 0and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, thesubstituent “n” shown in the preceding paragraph is 0. In otherembodiments, the substituent “n” shown in the preceding paragraph isless than 50, 40, 30, 20, 10, or 5.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In other embodiments, D and/or E in the compound of Formula I arefurther modified in order to facilitate cellular uptake. In someembodiments, lipidating or PEGylating a peptidomimetic macrocyclefacilitates cellular uptake, increases bioavailability, increases bloodcirculation, alters pharmacokinetics, decreases immunogenicity and/ordecreases the needed frequency of administration.

In other embodiments, at least one of [D] and [E] in the compound ofFormula I represents a moiety comprising an additionalmacrocycle-forming linker such that the peptidomimetic macrocyclecomprises at least two macrocycle-forming linkers. In a specificembodiment, a peptidomimetic macrocycle comprises two macrocycle-forminglinkers.

In the peptidomimetic macrocycles of the invention, any of themacrocycle-forming linkers described herein may be used in anycombination with any of the sequences shown in Tables 1-3 and also withany of the R-substituents indicated herein.

In some embodiments, the peptidomimetic macrocycle comprises at leastone α-helix motif. For example, A, B and/or C in the compound of FormulaI include one or more α-helices. As a general matter, α-helices includebetween 3 and 4 amino acid residues per turn. In some embodiments, theα-helix of the peptidomimetic macrocycle includes 1 to 5 turns and,therefore, 3 to 20 amino acid residues. In specific embodiments, theα-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In someembodiments, the macrocycle-forming linker stabilizes an α-helix motifincluded within the peptidomimetic macrocycle. Thus, in someembodiments, the length of the macrocycle-forming linker L from a firstCα to a second Ca is selected to increase the stability of an α-helix.In some embodiments, the macrocycle-forming linker spans from 1 turn to5 turns of the α-helix. In some embodiments, the macrocycle-forminglinker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turnsof the α-helix. In some embodiments, the length of themacrocycle-forming linker is approximately 5 Å to 9 Å per turn of theα-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelength is equal to approximately 5 carbon-carbon bonds to 13carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 2 turns of an α-helix, thelength is equal to approximately 8 carbon-carbon bonds to 16carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 3 turns of an α-helix, thelength is equal to approximately 14 carbon-carbon bonds to 22carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 4 turns of an α-helix, thelength is equal to approximately 20 carbon-carbon bonds to 28carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 5 turns of an α-helix, thelength is equal to approximately 26 carbon-carbon bonds to 34carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelinkage contains approximately 4 atoms to 12 atoms, approximately 6atoms to 10 atoms, or approximately 8 atoms. Where themacrocycle-forming linker spans approximately 2 turns of the α-helix,the linkage contains approximately 7 atoms to 15 atoms, approximately 9atoms to 13 atoms, or approximately 11 atoms. Where themacrocycle-forming linker spans approximately 3 turns of the α-helix,the linkage contains approximately 13 atoms to 21 atoms, approximately15 atoms to 19 atoms, or approximately 17 atoms. Where themacrocycle-forming linker spans approximately 4 turns of the α-helix,the linkage contains approximately 19 atoms to 27 atoms, approximately21 atoms to 25 atoms, or approximately 23 atoms. Where themacrocycle-forming linker spans approximately 5 turns of the α-helix,the linkage contains approximately 25 atoms to 33 atoms, approximately27 atoms to 31 atoms, or approximately 29 atoms. Where themacrocycle-forming linker spans approximately 1 turn of the α-helix, theresulting macrocycle forms a ring containing approximately 17 members to25 members, approximately 19 members to 23 members, or approximately 21members. Where the macrocycle-forming linker spans approximately 2 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 29 members to 37 members, approximately 31 members to 35members, or approximately 33 members. Where the macrocycle-forminglinker spans approximately 3 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 44 members to 52members, approximately 46 members to 50 members, or approximately 48members. Where the macrocycle-forming linker spans approximately 4 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 59 members to 67 members, approximately 61 members to 65members, or approximately 63 members. Where the macrocycle-forminglinker spans approximately 5 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 74 members to 82members, approximately 76 members to 80 members, or approximately 78members.

In some embodiments, L is a macrocycle-forming linker of the formula

Exemplary embodiments of such macrocycle-forming linkers L are shownbelow.

In other embodiments, the invention provides peptidomimetic macrocyclesof Formula (II) or (IIa):

wherein:

each A, C, D, and E is independently an amino acid;

B is an amino acid,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

and wherein A, B, C, D, and E, taken together with the crosslinked aminoacids connected by the macrocycle-forming linker -L₁-L₂-, form the aminoacid sequence of the peptidomimetic macrocycle which is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identicalto GHRH 1-44, GHRH 1-29 and/or to an amino acid sequence chosen from thegroup consisting of the amino acid sequences in Table 1, 2 or 4;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

v and w are independently integers from 1-1000, for example 1-100;

u, x, y and z are independently integers from 0-10, for example u is1-3; and

n is an integer from 1-5.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, the sum of x+y+z is at least 1. Inother embodiments of the invention, the sum of x+y+z is at least 2. Inother embodiments of the invention, the sum of x+y+z is 1, 2, 3, 4, 5,6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle ormacrocycle precursor of the invention is independently selected. Forexample, a sequence represented by the formula [A]_(x), when x is 3,encompasses embodiments where the amino acids are not identical, e.g.Gln-Asp-Ala as well as embodiments where the amino acids are identical,e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in theindicated ranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker-L₁-L₂- as measured from a first Cα to a second Cα is selected tostabilize a desired secondary peptide structure, such as an α-helixformed by residues of the peptidomimetic macrocycle including, but notnecessarily limited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker -L₁-L₂- are shownbelow.

Examples of peptidomimetic macrocycles of Formula (II) are shown inTable 4 and include SP-85, SP-86, SP-87, SP-88, SP-91, and SP-92.

Preparation of Peptidomimetic Macrocycles

Peptidomimetic macrocycles of the invention may be prepared by any of avariety of methods known in the art. For example, any of the residuesindicated by “X”, “Z” or “XX” in Tables 1, 2 or 4 may be substitutedwith a residue capable of forming a crosslinker with a second residue inthe same molecule or a precursor of such a residue.

Various methods to effect formation of peptidomimetic macrocycles areknown in the art. For example, the preparation of peptidomimeticmacrocycles of Formula I is described in Schafmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004);U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. Theα,α-disubstituted amino acids and amino acid precursors disclosed in thecited references may be employed in synthesis of the peptidomimeticmacrocycle precursor polypeptides. For example, the “S5-olefin aminoacid” is (S)-α-(2′-pentenyl) alanine and the “R8 olefin amino acid” is(R)-α-(2′-octenyl) alanine. Following incorporation of such amino acidsinto precursor polypeptides, the terminal olefins are reacted with ametathesis catalyst, leading to the formation of the peptidomimeticmacrocycle. In various embodiments, the following amino acids may beemployed in the synthesis of the peptidomimetic macrocycle:

In some embodiments, x+y+z is 3, and and A, B and C are independentlynatural or non-natural amino acids. In other embodiments, x+y+z is 6,and and A, B and C are independently natural or non-natural amino acids.

In some embodiments, the contacting step is performed in a solventselected from the group consisting of protic solvent, aqueous solvent,organic solvent, and mixtures thereof. For example, the solvent may bechosen from the group consisting of H₂O, THF, THF/H₂O, tBuOH/H₂O, DMF,DIPEA, CH₃CN or CH₂Cl₂, ClCH₂CH₂Cl or a mixture thereof. The solvent maybe a solvent which favors helix formation.

Alternative but equivalent protecting groups, leaving groups or reagentsare substituted, and certain of the synthetic steps are performed inalternative sequences or orders to produce the desired compounds.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein include, for example, those such as described inLarock, Comprehensive Organic Transformations, VCH Publishers (1989);Greene and Wuts, Protective Groups in Organic Synthesis, 2d. Ed., JohnWiley and Sons (1991); Fieser and Fieser, Fieser and Fieser's Reagentsfor Organic Synthesis, John Wiley and Sons (1994); and Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The peptidomimetic macrocycles disclosed herein are made, for example,by chemical synthesis methods, such as described in Fields et al.,Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H.Freeman & Co., New York, N.Y., 1992, p. 77. Hence, for example, peptidesare synthesized using the automated Merrifield techniques of solid phasesynthesis with the amine protected by either tBoc or Fmoc chemistryusing side chain protected amino acids on, for example, an automatedpeptide synthesizer (e.g., Applied Biosystems (Foster City, Calif.),Model 430A, 431, or 433).

One manner of producing the peptidomimetic precursors and peptidomimeticmacrocycles described herein uses solid phase peptide synthesis (SPPS).The C-terminal amino acid is attached to a cross-linked polystyreneresin via an acid labile bond with a linker molecule. This resin isinsoluble in the solvents used for synthesis, making it relativelysimple and fast to wash away excess reagents and by-products. TheN-terminus is protected with the Fmoc group, which is stable in acid,but removable by base. Side chain functional groups are protected asnecessary with base stable, acid labile groups.

Longer peptidomimetic precursors are produced, for example, byconjoining individual synthetic peptides using native chemical ligation.Alternatively, the longer synthetic peptides are biosynthesized by wellknown recombinant DNA and protein expression techniques. Such techniquesare provided in well-known standard manuals with detailed protocols. Toconstruct a gene encoding a peptidomimetic precursor of this invention,the amino acid sequence is reverse translated to obtain a nucleic acidsequence encoding the amino acid sequence, preferably with codons thatare optimum for the organism in which the gene is to be expressed. Next,a synthetic gene is made, typically by synthesizing oligonucleotideswhich encode the peptide and any regulatory elements, if necessary. Thesynthetic gene is inserted in a suitable cloning vector and transfectedinto a host cell. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. Thepeptide is purified and characterized by standard methods.

The peptidomimetic precursors are made, for example, in ahigh-throughput, combinatorial fashion using, for example, ahigh-throughput polychannel combinatorial synthesizer (e.g., ThuramedTETRAS multichannel peptide synthesizer from CreoSalus, Louisville, Ky.or Model Apex 396 multichannel peptide synthesizer from AAPPTEC, Inc.,Louisville, Ky.).

In some embodiments, the peptidomimetic macrocyles of the inventioncomprise triazole macrocycle-forming linkers. For example, the synthesisof such peptidomimetic macrocycles involves a multi-step process thatfeatures the synthesis of a peptidomimetic precursor containing an azidemoiety and an alkyne moiety; followed by contacting the peptidomimeticprecursor with a macrocyclization reagent to generate a triazole-linkedpeptidomimetic macrocycle. Such a process is described, for example, inU.S. application Ser. No. 12/037,041, filed on Feb. 25, 2008.Macrocycles or macrocycle precursors are synthesized, for example, bysolution phase or solid-phase methods, and can contain bothnaturally-occurring and non-naturally-occurring amino acids. See, forexample, Hunt, “The Non-Protein Amino Acids” in Chemistry andBiochemistry of the Amino Acids, edited by G. C. Barrett, Chapman andHall, 1985.

In some embodiments, an azide is linked to the α-carbon of a residue andan alkyne is attached to the α-carbon of another residue. In someembodiments, the azide moieties are azido-analogs of amino acidsL-lysine, D-lysine, alpha-methyl-L-lysine, alpha-methyl-D-lysine,L-ornithine, D-ornithine, alpha-methyl-L-omithine oralpha-methyl-D-ornithine. In another embodiment, the alkyne moiety isL-propargylglycine. In yet other embodiments, the alkyne moiety is anamino acid selected from the group consisting of L-propargylglycine,D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,(R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoicacid, (R)-2-amino-2-methyl-5-hexynoic acid,(S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoicacid, (S)-2-amino-2-methyl-7-octynoic acid,(R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoicacid and (R)-2-amino-2-methyl-8-nonynoic acid.

The following synthetic schemes are provided solely to illustrate thepresent invention and are not intended to limit the scope of theinvention, as described herein. To simplify the drawings, theillustrative schemes depict azido amino acid analogsε-azido-α-methyl-L-lysine and ε-azido-α-methyl-D-lysine, and alkyneamino acid analogs L-propargylglycine, (S)-2-amino-2-methyl-4-pentynoicacid, and (S)-2-amino-2-methyl-6-heptynoic acid. Thus, in the followingsynthetic schemes, each R₁, R₂, R₇ and R₈ is —H; each L₁ is —(CH₂)₄—;and each L₂ is —(CH₂)—. However, as noted throughout the detaileddescription above, many other amino acid analogs can be employed inwhich R₁, R₂, R₇, R₈, L₁ and L₂ can be independently selected from thevarious structures disclosed herein.

Synthetic Scheme 1 describes the preparation of several compounds of theinvention. Ni(II) complexes of Schiff bases derived from the chiralauxiliary (S)-2-[N—(N′-benzylprolyl)amino]benzophenone (BPB) and aminoacids such as glycine or alanine are prepared as described in Belokon etal. (1998), Tetrahedron Asymm. 9:4249-4252. The resulting complexes aresubsequently reacted with alkylating reagents comprising an azido oralkynyl moiety to yield enantiomerically enriched compounds of theinvention. If desired, the resulting compounds can be protected for usein peptide synthesis.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 2, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solution-phaseor solid-phase peptide synthesis (SPPS) using the commercially availableamino acid N-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected formsof the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA). The peptidomimetic precursor is reacted as a crude mixture or ispurified prior to reaction with a macrocyclization reagent such as aCu(I) in organic or aqueous solutions (Rostovtsev et al. (2002), Angew.Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002), J Org. Chem.67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc. 125:11782-11783;Punna et al. (2005), Angew. Chem. Int. Ed. 44:2215-2220). In oneembodiment, the triazole forming reaction is performed under conditionsthat favor α-helix formation. In one embodiment, the macrocyclizationstep is performed in a solvent chosen from the group consisting of H₂O,THF, CH₃CN, DMF, DIPEA, tBuOH or a mixture thereof. In anotherembodiment, the macrocyclization step is performed in DMF. In someembodiments, the macrocyclization step is performed in a bufferedaqueous or partially aqueous solvent.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 3, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solid-phasepeptide synthesis (SPPS) using the commercially available amino acidN-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected forms of theamino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is reacted with a macrocyclization reagent suchas a Cu(I) reagent on the resin as a crude mixture (Rostovtsev et al.(2002), Angew. Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002), J Org.Chem. 67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc.125:11782-11783; Punna et al. (2005), Angew. Chem. Int. Ed.44:2215-2220). The resultant triazole-containing peptidomimeticmacrocycle is then deprotected and cleaved from the solid-phase resin bystandard conditions (e.g., strong acid such as 95% TFA). In someembodiments, the macrocyclization step is performed in a solvent chosenfrom the group consisting of CH₂Cl₂, ClCH₂CH₂Cl, DMF, THF, NMP, DIPEA,2,6-lutidine, pyridine, DMSO, H₂O or a mixture thereof. In someembodiments, the macrocyclization step is performed in a bufferedaqueous or partially aqueous solvent.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 4, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solution-phaseor solid-phase peptide synthesis (SPPS) using the commercially availableamino acid N-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected formsof the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA). The peptidomimetic precursor is reacted as a crude mixture or ispurified prior to reaction with a macrocyclization reagent such as aRu(II) reagents, for example Cp*RuCl(PPh₃)₂ or [Cp*RuCl]₄ (Rasmussen etal. (2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem.Soc. 127:15998-15999). In some embodiments, the macrocyclization step isperformed in a solvent chosen from the group consisting of DMF, CH₃CNand THF.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 5, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solid-phasepeptide synthesis (SPPS) using the commercially available amino acidN-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected forms of theamino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is reacted with a macrocyclization reagent suchas a Ru(II) reagent on the resin as a crude mixture. For example, thereagent can be Cp*RuCl(PPh₃)₂ or [Cp*RuCl]₄ (Rasmussen et al. (2007),Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem. Soc.127:15998-15999). In some embodiments, the macrocyclization step isperformed in a solvent chosen from the group consisting of CH₂Cl₂,ClCH₂CH₂Cl, CH₃CN, DMF, and THF.

The present invention contemplates the use of non-naturally-occurringamino acids and amino acid analogs in the synthesis of thepeptidomimetic macrocycles described herein. Any amino acid or aminoacid analog amenable to the synthetic methods employed for the synthesisof stable triazole containing peptidomimetic macrocycles can be used inthe present invention. For example, L-propargylglycine is contemplatedas a useful amino acid in the present invention. However, otheralkyne-containing amino acids that contain a different amino acid sidechain are also useful in the invention. For example, L-propargylglycinecontains one methylene unit between the α-carbon of the amino acid andthe alkyne of the amino acid side chain. The invention also contemplatesthe use of amino acids with multiple methylene units between theα-carbon and the alkyne. Also, the azido-analogs of amino acidsL-lysine, D-lysine, alpha-methyl-L-lysine, and alpha-methyl-D-lysine arecontemplated as useful amino acids in the present invention. However,other terminal azide amino acids that contain a different amino acidside chain are also useful in the invention. For example, theazido-analog of L-lysine contains four methylene units between theα-carbon of the amino acid and the terminal azide of the amino acid sidechain. The invention also contemplates the use of amino acids with fewerthan or greater than four methylene units between the α-carbon and theterminal azide. Table 3 shows some amino acids useful in the preparationof peptidomimetic macrocycles disclosed herein.

TABLE 3

N-α-Fmoc-L-propargyl glycine

N-α-Fmoc-(S)-2-amino-2- methyl-4-pentynoic acid

N-α-Fmoc-(S)-2-amino-2- methyl-5-hexynoic acid

N-α-Fmoc-(S)-2-amino-2- methyl-6-heptynoic acid

N-α-Fmoc-(S)-2-amino-2- methyl-7-octynoic acid

N-α-Fmoc-(S)-2-amino-2- methyl-8-nonynoic acid

N-α-Fmoc-D-propargyl glycine

N-α-Fmoc-(R)-2-amino-2- methyl-4-pentynoic acid

N-α-Fmoc-(R)-2-amino-2- methyl-5-hexynoic acid

N-α-Fmoc-(R)-2-amino-2- methyl-6-heptynoic acid

N-α-Fmoc-(R)-2-amino-2- methyl-7-octynoic acid

N-α-Fmoc-(R)-2-amino-2- methyl-8-nonynoic acid

N-α-Fmoc-ϵ-azido-L-lysine

N-α-Fmoc-ϵ-azido- α-methyl-L-lysine

N-α-Fmoc-δ- azido-L-ornithine

N-α-Fmoc-δ-azido-α- methyl-L-ornithine

N-α-Fmoc-ϵ-azido-D-Iysine

N-α-Fmoc-ϵ-azido-α- methyl-D-lysine

N-α-Fmoc-δ-azido- D-ornithine

N-α-Fmoc-ϵ-azido-α- methyl-D-ornithine

-   -   Table 3 shows exemplary amino acids useful in the preparation of        peptidomimetic macrocycles disclosed herein.

In some embodiments the amino acids and amino acid analogs are of theD-configuration. In other embodiments they are of the L-configuration.In some embodiments, some of the amino acids and amino acid analogscontained in the peptidomimetic are of the D-configuration while some ofthe amino acids and amino acid analogs are of the L-configuration. Insome embodiments the amino acid analogs are α,α-disubstituted, such asα-methyl-L-propargylglycine, α-methyl-D-propargylglycine,ε-azido-alpha-methyl-L-lysine, and ε-azido-alpha-methyl-D-lysine. Insome embodiments the amino acid analogs are N-alkylated, e.g.,N-methyl-L-propargylglycine, N-methyl-D-propargylglycine,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine.

In some embodiments, the —NH moiety of the amino acid is protected usinga protecting group, including without limitation -Fmoc and -Boc. Inother embodiments, the amino acid is not protected prior to synthesis ofthe peptidomimetic macrocycle.

Additional methods of forming peptidomimetic macrocycles which areenvisioned as suitable to perform the present invention include thosedisclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem (2003), 68,pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp.1403-1406; U.S. Pat. Nos. 5,364,851; 5,446,128; 5,824,483; 6,713,280;and 7,202,332. In such embodiments, amino acid precursors are usedcontaining an additional substituent R— at the alpha position. Suchaminoacids are incorporated into the macrocycle precursor at the desiredpositions, which may be at the positions where the crosslinker issubstituted or, alternatively, elsewhere in the sequence of themacrocycle precursor. Cyclization of the precursor is then performedaccording to the indicated method.

For example, a peptidomimetic macrocycle of Formula (II) is prepared asindicated:

-   -   wherein each AA1, AA2, AA3 is independently an amino acid side        chain.

In other embodiments, a peptidomimetic macrocycle of Formula (II) isprepared as indicated:

In some embodiments, a peptidomimetic macrocycle is obtained in morethan one isomer, for example due to the configuration of a double bondwithin the structure of the crosslinker (E vs Z). Such isomers can orcan not be separable by conventional chromatographic methods. In someembodiments, one isomer has improved biological properties relative tothe other isomer. In one embodiment, an E crosslinker olefin isomer of apeptidomimetic macrocycle has better solubility, better target affinity,better in vivo or in vitro efficacy, higher helicity, or improved cellpermeability relative to its Z counterpart. In another embodiment, a Zcrosslinker olefin isomer of a peptidomimetic macrocycle has bettersolubility, better target affinity, better in vivo or in vitro efficacy,higher helicity, or improved cell permeability relative to its Ecounterpart.

Assays

The properties of the peptidomimetic macrocycles of the invention areassayed, for example, by using the methods described below. In someembodiments, a peptidomimetic macrocycle of the invention has improvedbiological properties relative to a corresponding polypeptide lackingthe substituents described herein.

Assay to Determine α-Helicity.

In solution, the secondary structure of polypeptides with α-helicaldomains will reach a dynamic equilibrium between random coil structuresand α-helical structures, often expressed as a “percent helicity”. Thus,for example, alpha-helical domains are predominantly random coils insolution, with α-helical content usually under 25%. Peptidomimeticmacrocycles with optimized linkers, on the other hand, possess, forexample, an alpha-helicity that is at least two-fold greater than thatof a corresponding uncrosslinked polypeptide. In some embodiments,macrocycles of the invention will possess an alpha-helicity of greaterthan 50%. To assay the helicity of peptidomimetic macrocyles of theinvention, the compounds are dissolved in an aqueous solution (e.g. 50mM potassium phosphate solution at pH 7, or distilled H₂O, toconcentrations of 25-50 μM). Circular dichroism (CD) spectra areobtained on a spectropolarimeter (e.g., Jasco J-710) using standardmeasurement parameters (e.g. temperature, 20° C.; wavelength, 190-260nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10;response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helicalcontent of each peptide is calculated by dividing the mean residueellipticity (e.g. [Φ]222obs) by the reported value for a model helicaldecapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm).

A peptidomimetic macrocycle of the invention comprising a secondarystructure such as an α-helix exhibits, for example, a higher meltingtemperature than a corresponding uncrosslinked polypeptide. Typicallypeptidomimetic macrocycles of the invention exhibit Tm of >60° C.representing a highly stable structure in aqueous solutions. To assaythe effect of macrocycle formation on meltine temperature,peptidomimetic macrocycles or unmodified peptides are dissolved indistilled H₂O (e.g. at a final concentration of 50 μM) and the Tm isdetermined by measuring the change in ellipticity over a temperaturerange (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710)using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1nm; temperature increase rate: 1° C./min; path length, 0.1 cm).

Protease Resistance Assay.

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, typically buriesthe amide backbone and therefore may shield it from proteolyticcleavage. The peptidomimetic macrocycles of the present invention may besubjected to in vitro trypsin proteolysis to assess for any change indegradation rate compared to a corresponding uncrosslinked polypeptide.For example, the peptidomimetic macrocycle and a correspondinguncrosslinked polypeptide are incubated with trypsin agarose and thereactions quenched at various time points by centrifugation andsubsequent HPLC injection to quantitate the residual substrate byultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycleand peptidomimetic precursor (5 mcg) are incubated with trypsin agarose(Pierce) (S/E˜125) for 0, 10, 20, 90, and 180 minutes. Reactions arequenched by tabletop centrifugation at high speed; remaining substratein the isolated supernatant is quantified by HPLC-based peak detectionat 280 nm. The proteolytic reaction displays first order kinetics andthe rate constant, k, is determined from a plot of ln [S] versus time(k=−1×slope).

Ex Vivo Stability Assay.

Peptidomimetic macrocycles with optimized linkers possess, for example,an ex vivo half-life that is at least two-fold greater than that of acorresponding uncrosslinked polypeptide, and possess an ex vivohalf-life of 12 hours or more. For ex vivo serum stability studies, avariety of assays may be used. For example, a peptidomimetic macrocycleand a corresponding uncrosslinked polypeptide (2 mcg) are incubated withfresh mouse, rat and/or human serum (2 mL) at 37° C. for 0, 1, 2, 4, 8,and 24 hours. To determine the level of intact compound, the followingprocedure may be used: The samples are extracted by transferring 100 μlof sera to 2 ml centrifuge tubes followed by the addition of 10 μL of50% formic acid and 500 μL acetonitrile and centrifugation at 14,000 RPMfor 10 min at 4±2° C. The supernatants are then transferred to fresh 2ml tubes and evaporated on Turbovap under N₂<10 psi, 37° C. The samplesare reconstituted in 100 μL of 50:50 acetonitrile:water and submitted toLC-MS/MS analysis.

In Vitro Binding Assays.

To assess the binding and affinity of peptidomimetic macrocycles andpeptidomimetic precursors to acceptor proteins, a fluorescencepolarization assay (FPA) issued, for example. The FPA technique measuresthe molecular orientation and mobility using polarized light andfluorescent tracer. When excited with polarized light, fluorescenttracers (e.g., FITC) attached to molecules with high apparent molecularweights (e.g. FITC-labeled peptides bound to a large protein) emithigher levels of polarized fluorescence due to their slower rates ofrotation as compared to fluorescent tracers attached to smallermolecules (e.g. FITC-labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) areincubated with the acceptor protein (25-1000 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.Binding activity is measured, for example, by fluorescence polarizationon a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd valuesmay be determined by nonlinear regression analysis using, for example,Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). Apeptidomimetic macrocycle of the invention shows, in some instances,similar or lower Kd than a corresponding uncrosslinked polypeptide.

In Vitro Displacement Assays to Characterize Antagonists ofPeptide-Protein Interactions.

To assess the binding and affinity of compounds that antagonize theinteraction between a peptide and an acceptor protein, a fluorescencepolarization assay (FPA) utilizing a fluoresceinated peptidomimeticmacrocycle derived from a peptidomimetic precursor sequence is used, forexample. The FPA technique measures the molecular orientation andmobility using polarized light and fluorescent tracer. When excited withpolarized light, fluorescent tracers (e.g., FITC) attached to moleculeswith high apparent molecular weights (e.g. FITC-labeled peptides boundto a large protein) emit higher levels of polarized fluorescence due totheir slower rates of rotation as compared to fluorescent tracersattached to smaller molecules (e.g. FITC-labeled peptides that are freein solution). A compound that antagonizes the interaction between thefluoresceinated peptidomimetic macrocycle and an acceptor protein willbe detected in a competitive binding FPA experiment.

For example, putative antagonist compounds (1 nM to 1 mM) and afluoresceinated peptidomimetic macrocycle (25 nM) are incubated with theacceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL,pH 7.4) for 30 minutes at room temperature. Antagonist binding activityis measured, for example, by fluorescence polarization on a luminescencespectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determinedby nonlinear regression analysis using, for example, Graphpad Prismsoftware (GraphPad Software, Inc., San Diego, Calif.).

Any class of molecule, such as small organic molecules, peptides,oligonucleotides or proteins can be examined as putative antagonists inthis assay.

Assay for Protein-Ligand Binding by Affinity Selection—Mass Spectrometry

To assess the binding and affinity of test compounds for proteins, anaffinity-selection mass spectrometry assay is used, for example.Protein-ligand binding experiments are conducted according to thefollowing representative procedure outlined for a system-wide controlexperiment using 1 μM peptidomimetic macrocycle plus 5 μM targetprotein. A 1 μL DMSO aliquot of a 40 μM stock solution of peptidomimeticmacrocycle is dissolved in 19 μL of PBS (Phosphate-buffered saline: 50mM, pH 7.5 Phosphate buffer containing 150 mM NaCl). The resultingsolution is mixed by repeated pipetting and clarified by centrifugationat 10 000 g for 10 min. To a 4 μL aliquot of the resulting supernatantis added 4 μL of 10 μM target protein in PBS. Each 8.0 μL experimentalsample thus contains 40 pmol (1.5 μg) of protein at 5.0 μM concentrationin PBS plus 1 μM peptidomimetic macrocycle and 2.5% DMSO. Duplicatesamples thus prepared for each concentration point are incubated for 60min at room temperature, and then chilled to 4° C. prior tosize-exclusion chromatography-LC-MS analysis of 5.0 μL injections.Samples containing a target protein, protein-ligand complexes, andunbound compounds are injected onto an SEC column, where the complexesare separated from non-binding component by a rapid SEC step. The SECcolumn eluate is monitored using UV detectors to confirm that theearly-eluting protein fraction, which elutes in the void volume of theSEC column, is well resolved from unbound components that are retainedon the column. After the peak containing the protein and protein-ligandcomplexes elutes from the primary UV detector, it enters a sample loopwhere it is excised from the flow stream of the SEC stage andtransferred directly to the LC-MS via a valving mechanism. The (M+3H)³⁺ion of the peptidomimetic macrocycle is observed by ESI-MS at theexpected m/z, confirming the detection of the protein-ligand complex.

Assay for Protein-Ligand Kd Titration Experiments.

To assess the binding and affinity of test compounds for proteins, aprotein-ligand Kd titration experiment is performed, for example.Protein-ligand K_(d) titrations experiments are conducted as follows: 2μL DMSO aliquots of a serially diluted stock solution of titrantpeptidomimetic macrocycle (5, 2.5, . . . , 0.098 mM) are prepared thendissolved in 38 μL of PBS. The resulting solutions are mixed by repeatedpipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0μL aliquots of the resulting supernatants is added 4.0 μL of 10 μMtarget protein in PBS. Each 8.0 μL experimental sample thus contains 40pmol (1.5 μg) of protein at 5.0 μM concentration in PBS, varyingconcentrations (125, 62.5, . . . , 0.24 μM) of the titrant peptide, and2.5% DMSO. Duplicate samples thus prepared for each concentration pointare incubated at room temperature for 30 min, then chilled to 4° C.prior to SEC-LC-MS analysis of 2.0 μL injections. The (M+H)¹⁺, (M+2H)²⁺,(M+3H)³⁺, and/or (M+Na)¹⁺ ion is observed by ESI-MS; extracted ionchromatograms are quantified, then fit to equations to derive thebinding affinity K_(d) as described in “A General Technique to RankProtein-Ligand Binding Affinities and Determine Allosteric vs. DirectBinding Site Competition in Compound Mixtures.” Annis, D. A.; Nazef, N.;Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126,15495-15503; also in “ALIS: An Affinity Selection-Mass SpectrometrySystem for the Discovery and Characterization of Protein-LigandInteractions” D. A. Annis, C.-C. Chuang, and N. Nazef. In MassSpectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G:Wiley-VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (SeriesEditors): Methods and Principles in Medicinal Chemistry.

Assay for Competitive Binding Experiments by Affinity Selection-MassSpectrometry

To determine the ability of test compounds to bind competitively toproteins, an affinity selection mass spectrometry assay is performed,for example. A mixture of ligands at 40 μM per component is prepared bycombining 2 μL aliquots of 400 μM stocks of each of the three compoundswith 14 μL of DMSO. Then, 1 μL aliquots of this 40 μM per componentmixture are combined with 1 μL DMSO aliquots of a serially diluted stocksolution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . , 0.078mM). These 2 L samples are dissolved in 38 μL of PBS. The resultingsolutions were mixed by repeated pipetting and clarified bycentrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of theresulting supernatants is added 4.0 μL of 10 μM target protein in PBS.Each 8.0 μL experimental sample thus contains 40 pmol (1.5 μg) ofprotein at 5.0 μM concentration in PBS plus 0.5 μM ligand, 2.5% DMSO,and varying concentrations (125, 62.5, . . . , 0.98 μM) of the titrantpeptidomimetic macrocycle. Duplicate samples thus prepared for eachconcentration point are incubated at room temperature for 60 min, thenchilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections.Additional details on these and other methods are provided in “A GeneralTechnique to Rank Protein-Ligand Binding Affinities and DetermineAllosteric vs. Direct Binding Site Competition in Compound Mixtures.”Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am.Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An AffinitySelection-Mass Spectrometry System for the Discovery andCharacterization of Protein-Ligand Interactions” D. A. Annis, C.-C.Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry.Edited by Wanner K, Hofner G: Wiley-VCH; 2007:121-184. Mannhold R,Kubinyi H, Folkers G (Series Editors): Methods and Principles inMedicinal Chemistry.

Binding Assays in Intact Cells.

It is possible to measure binding of peptides or peptidomimeticmacrocycles to their natural acceptors in intact cells byimmunoprecipitation experiments. For example, intact cells are incubatedwith fluoresceinated (FITC-labeled) compounds for 4 hrs in the absenceof serum, followed by serum replacement and further incubation thatranges from 4-18 hrs. Cells are then pelleted and incubated in lysisbuffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and proteaseinhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at14,000 rpm for 15 minutes and supernatants collected and incubated with10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed byfurther 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of50% bead slurry). After quick centrifugation, the pellets are washed inlysis buffer containing increasing salt concentration (e.g., 150, 300,500 mM). The beads are then re-equilibrated at 150 mM NaCl beforeaddition of SDS-containing sample buffer and boiling. Aftercentrifugation, the supernatants are optionally electrophoresed using4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-Pmembranes. After blocking, blots are optionally incubated with anantibody that detects FITC and also with one or more antibodies thatdetect proteins that bind to the peptidomimetic macrocycle.

Cellular Penetrability Assays.

To measure the cell penetrability of peptidomimetic macrocycles andcorresponding uncrosslinked macrocycle, intact cells are incubated withfluoresceinated peptidomimetic macrocycles or correspondinguncrosslinked macrocycle (10 μM) for 4 hrs in serum free media at 37°C., washed twice with media and incubated with trypsin (0.25%) for 10min at 37° C. The cells are washed again and resuspended in PBS.Cellular fluorescence is analyzed, for example, by using either aFACSCalibur flow cytometer or Cellomics' KineticScan® HCS Reader.

In Vivo Stability Assay.

To investigate the in vivo stability of the peptidomimetic macrocycles,the compounds are, for example, administered to mice and/or rats by IV,IP, PO or inhalation routes at concentrations ranging from 0.1 to 50mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8hrs and 24 hours post-injection. Levels of intact compound in 25 μL offresh serum are then measured by LC-MS/MS as above.

Clinical Trials.

To determine the suitability of the peptidomimetic macrocycles of theinvention for treatment of humans, clinical trials are performed. Forexample, patients diagnosed with a muscle wasting disease orlipodystrophy and in need of treatment are selected and separated intreatment and one or more control groups, wherein the treatment group isadministered a peptidomimetic macrocycle of the invention, while thecontrol groups receive a placebo or a known GHRH or GH drug. Thetreatment safety and efficacy of the peptidomimetic macrocycles of theinvention can thus be evaluated by performing comparisons of the patientgroups with respect to factors such as survival and quality-of-life. Inthis example, the patient group treated with a peptidomimetic macrocycleshow improved long-term survival compared to a patient control grouptreated with a placebo.

Pharmaceutical Compositions and Routes of Administration

The peptidomimetic macrocycles of the invention also includepharmaceutically acceptable derivatives or prodrugs thereof. A“pharmaceutically acceptable derivative” means any pharmaceuticallyacceptable salt, ester, salt of an ester, pro-drug or other derivativeof a compound of this invention which, upon administration to arecipient, is capable of providing (directly or indirectly) a compoundof this invention. Particularly favored pharmaceutically acceptablederivatives are those that increase the bioavailability of the compoundsof the invention when administered to a mammal (e.g., by increasingabsorption into the blood of an orally administered compound) or whichincreases delivery of the active compound to a biological compartment(e.g., the brain or lymphatic system) relative to the parent species.Some pharmaceutically acceptable derivatives include a chemical groupwhich increases aqueous solubility or active transport across thegastrointestinal mucosa.

In some embodiments, the peptidomimetic macrocycles of the invention aremodified by covalently or non-covalently joining appropriate functionalgroups to enhance selective biological properties. Such modificationsinclude those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism, and alter rate ofexcretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers include eithersolid or liquid carriers. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. A solid carrier can be one or more substances, which also actsas diluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

Suitable solid excipients are carbohydrate or protein fillers include,but are not limited to sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; and gums including arabic and tragacanth;as well as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents are added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

When the compositions of this invention comprise a combination of apeptidomimetic macrocycle and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 1 to 100%, and morepreferably between about 5 to 95% of the dosage normally administered ina monotherapy regimen. In some embodiments, the additional agents areadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents are part of asingle dosage form, mixed together with the compounds of this inventionin a single composition.

In some embodiments, the compositions are present as unit dosage formsthat can deliver, for example, from about 0.0001 mg to about 1,000 mg ofthe peptidomimetic macrocycles, salts thereof, prodrugs thereof,derivatives thereof, or any combination of these. Thus, the unit dosageforms can deliver, for example, in some embodiments, from about 1 mg toabout 900 mg, from about 1 mg to about 800 mg, from about 1 mg to about700 mg, from about 1 mg to about 600 mg, from about 1 mg to about 500mg, from about 1 mg to about 400 mg, from about 1 mg to about 300 mg,from about 1 mg to about 200 mg, from about 1 mg to about 100 mg, fromabout 1 mg to about 10 mg, from about 1 mg to about 5 mg, from about 0.1mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 10 mg toabout 1,000 mg, from about 50 mg to about 1,000 mg, from about 100 mg toabout 1,000 mg, from about 200 mg to about 1,000 mg, from about 300 mgto about 1,000 mg, from about 400 mg to about 1,000 mg, from about 500mg to about 1,000 mg, from about 600 mg to about 1,000 mg, from about700 mg to about 1,000 mg, from about 800 mg to about 1,000 mg, fromabout 900 mg to about 1,000 mg, from about 10 mg to about 900 mg, fromabout 100 mg to about 800 mg, from about 200 mg to about 700 mg, or fromabout 300 mg to about 600 mg of the peptidomimetic macrocycles, saltsthereof, prodrugs thereof, derivatives thereof, or any combination ofthese.

In some embodiments, the compositions are present as unit dosage formsthat can deliver, for example, about 1 mg, about 2 mg, about 3 mg, about4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg,about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about500 mg, about 600 mg, about 700 mg, about 800 mg, or about 800 mg ofpeptidomimetic macrocycles, salts thereof, prodrugs thereof, derivativesthereof, or any combination of these.

Suitable routes of administration include, but are not limited to, oral,intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary,transmucosal, transdermal, vaginal, otic, nasal, and topicaladministration. In addition, by way of example only, parenteral deliveryincludes intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intralymphatic, and intranasal injections.

In certain embodiments, a composition as described herein isadministered in a local rather than systemic manner, for example, viainjection of the compound directly into an organ. In specificembodiments, long acting formulations are administered by implantation(for example subcutaneously or intramuscularly) or by intramuscularinjection. Furthermore, in other embodiments, the drug is delivered in atargeted drug delivery system, for example, in a liposome coated withorgan-specific antibody. In such embodiments, the liposomes are targetedto and taken up selectively by the organ. In yet other embodiments, thecompound as described herein is provided in the form of a rapid releaseformulation, in the form of an extended release formulation, or in theform of an intermediate release formulation. In yet other embodiments,the compound described herein is administered topically.

In another embodiment, compositions described herein are formulated fororal administration. Compositions described herein are formulated bycombining a peptidomimetic macrocycle with, e.g., pharmaceuticallyacceptable carriers or excipients. In various embodiments, the compoundsdescribed herein are formulated in oral dosage forms that include, byway of example only, tablets, powders, pills, dragees, capsules,liquids, gels, syrups, elixirs, slurries, suspensions and the like.

In certain embodiments, pharmaceutical preparations for oral use areobtained by mixing one or more solid excipient with one or more of thepeptidomimetic macrocycles described herein, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as: for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methylcellulose,microcrystalline cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP orpovidone) or calcium phosphate. In specific embodiments, disintegratingagents are optionally added. Disintegrating agents include, by way ofexample only, cross-linked croscarmellose sodium, polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

In one embodiment, dosage forms, such as dragee cores and tablets, areprovided with one or more suitable coating. In specific embodiments,concentrated sugar solutions are used for coating the dosage form. Thesugar solutions, optionally contain additional components, such as byway of example only, gum arabic, talc, polyvinylpyrrolidone, carbopolgel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,and suitable organic solvents or solvent mixtures. Dyestuffs and/orpigments are also optionally added to the coatings for identificationpurposes. Additionally, the dyestuffs and/or pigments are optionallyutilized to characterize different combinations of active compounddoses.

In certain embodiments, therapeutically effective amounts of at leastone of the peptidomimetic macrocycles described herein are formulatedinto other oral dosage forms. Oral dosage forms include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. In specificembodiments, push-fit capsules contain the active ingredients inadmixture with one or more filler. Fillers include, by way of exampleonly, lactose, binders such as starches, and/or lubricants such as talcor magnesium stearate and, optionally, stabilizers. In otherembodiments, soft capsules, contain one or more active compound that isdissolved or suspended in a suitable liquid. Suitable liquids include,by way of example only, one or more fatty oil, liquid paraffin, orliquid polyethylene glycol. In addition, stabilizers are optionallyadded.

In other embodiments, therapeutically effective amounts of at least oneof the peptidomimetic macrocycles described herein are formulated forbuccal or sublingual administration. Formulations suitable for buccal orsublingual administration include, by way of example only, tablets,lozenges, or gels. In still other embodiments, the peptidomimeticmacrocycles described herein are formulated for parenertal injection,including formulations suitable for bolus injection or continuousinfusion. In specific embodiments, formulations for injection arepresented in unit dosage form (e.g., in ampoules) or in multi-dosecontainers. Preservatives are, optionally, added to the injectionformulations. In still other embodiments, pharmaceutical compositionsare formulated in a form suitable for parenteral injection as a sterilesuspensions, solutions or emulsions in oily or aqueous vehicles.Parenteral injection formulations optionally contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. In specificembodiments, pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.In additional embodiments, suspensions of the active compounds areprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles for use in the pharmaceutical compositionsdescribed herein include, by way of example only, fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. In certain specific embodiments, aqueousinjection suspensions contain substances which increase the viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension contains suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions. Alternatively, in otherembodiments, the active ingredient is in powder form for constitutionwith a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Pharmaceutical compositions herein can be administered, for example,once or twice or three or four or five or six times per day, or once ortwice or three or four or five or six times per week, and can beadministered, for example, for a day, a week, a month, 3 months, sixmonths, a year, five years, or for example ten years.

Methods of Use

In one aspect, the present invention provides novel peptidomimeticmacrocycles that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the peptidomimetic macrocycles are modeled. For example, inthe GHRH system, labeled peptidomimetic macrocycles based on GHRH can beused in a binding assay along with small molecules that competitivelybind to the GHRH receptor. Competitive binding studies allow for rapidin vitro evaluation and determination of drug candidates specific forthe GHRH system. Such binding studies may be performed with any of thepeptidomimetic macrocycles disclosed herein and their binding partners.

The invention further provides for the generation of antibodies againstthe peptidomimetic macrocycles. In some embodiments, these antibodiesspecifically bind both the peptidomimetic macrocycle and the precursorpeptides, such as GHRH, to which the peptidomimetic macrocycles arerelated. Such antibodies, for example, disrupt the nativeprotein-protein interactions, for example, between GHRH and the GHRHreceptor.

In another aspect, the present invention provides methods to activatethe GHRH receptor, thereby stimulating production and release of growthhormone, which in turn can increase lean muscle mass or reduce adiposetissue, for example visceral and/or abdominal adipose tissue. In someembodiments, subject suffering from obesity, for example abdominalobesity, are treated using pharmaceutical compositions of the invention.See, e.g. Makimura et al., J. Clin. Endocrinol. Metab. 2009, 94(12):5131-5138, which is hereby incorporated by reference.

In yet another aspect, the present invention provides methods fortreating muscle wasting diseases that include anorexias, cachexias (suchas cancer cachexia, chronic heart failure cachexia, chronic obstructivepulmonary disease cachexia, rheumatoid arthritis cachexia) andsarcopenias, methods for treating lipodystrophies that include HIVlipodystrophy, methods for treating growth hormone disorders thatinclude adult and pediatric growth hormone deficiencies, or methods fortreating gastroparesis or short bowel syndrome. These methods compriseadministering an effective amount of a compound of the invention to awarm blooded animal, including a human. In some embodiments, apharmaceutical composition provided herein used in the treatment ofmuscle wasting diseases is administered no more frequently than oncedaily, no more frequently than every other day, no more frequently thantwice weekly, no more frequently than weekly, or no more frequently thanevery other week.

In some embodiments, provided herein are methods for treating adultgrowth hormone deficiencies. Such deficiencies may be cause, forexample, by damage or injury to the pituitary gland or the hypothalamus.Frequently, adult-onset growth hormone deficiency is caused by pituitarytumors or treatment of such tumors, for example by cranial irradiation.Adult growth hormone deficiency may also be caused by a reduced bloodsupply to the pituitary gland. In some embodiments, a pharmaceuticalcomposition of the invention used in treatment of adult growth hormonedeficiency is administered no more frequently than once daily, no morefrequently than every other day, no more frequently than twice weekly,no more frequently than weekly, or no more frequently than every otherweek.

In some embodiments, provided herein are methods for treating pediatricgrowth hormone deficiencies. Growth hormone deficiency in children isoften idiophathic. However, possible causes include mutations in genesincluding GHRHR or GH1, congenital malformations involving the pituitary(such as septo-optic dysplasia or posterior pituitary ectopia), chronickidney disease, intracranial tumors (e.g. in or near the sella turcica,such as craniopharyngioma), damage to the pituitary from radiationtherapy to the cranium (for cancers such as leukemia or brain tumors),surgery, trauma or intracranial disease (e.g. hydrocephalus), autoimmuneinflammation (hypophysitis), ischemic or hemorrhagic infarction from lowblood pressure (Sheehan syndrome) or hemorrhage pituitary apoplexy.Growth hormone deficiency is observed in congenital diseases such asPrader-Willi syndrome, Turner syndrome, or short stature homeobox gene(SHOX) deficiency, idiopathic short stature, or in infants who are smallfor gestational age. In some embodiments, a composition of the inventionused in treatment of pediatric growth hormone deficiency is administeredno more frequently than once daily, no more frequently than every otherday, no more frequently than twice weekly, no more frequently thanweekly, or no more frequently than every other week.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

In some embodiments, the invention provides peptidomimetic macrocyclesand methods of use as described in the items below.

Item 1. A peptidomimetic macrocycle comprising an amino acid sequencewhich is at least about 60% identical to an amino acid sequence chosenfrom the group consisting of the amino acid sequences in Tables 1, 2 or4.

Item 2. The peptidomimetic macrocycle of item 1, wherein the amino acidsequence of said peptidomimetic macrocycle is at least about 80%identical to an amino acid sequence chosen from the group consisting ofthe amino acid sequences in Tables 1, 2 or 4.

Item 3. The peptidomimetic macrocycle of item 1, wherein the amino acidsequence of said peptidomimetic macrocycle is at least about 90%identical to an amino acid sequence chosen from the group consisting ofthe amino acid sequences in Tables 1, 2 or 4.

Item 4. The peptidomimetic macrocycle of item 1, wherein the amino acidsequence of said peptidomimetic macrocycle is chosen from the groupconsisting of the amino acid sequences in Tables 1, 2 or 4.

Item 5. The peptidomimetic macrocycle of item 1, wherein thepeptidomimetic macrocycle comprises a helix.

Item 6. The peptidomimetic macrocycle of item 1, wherein thepeptidomimetic macrocycle comprises an α-helix.

Item 7. The peptidomimetic macrocycle of item 1, wherein thepeptidomimetic macrocycle comprises an α,α-disubstituted amino acid.

Item 8. The peptidomimetic macrocycle of item 1, wherein thepeptidomimetic macrocycle comprises a crosslinker linking theα-positions of at least two amino acids.

Item 9. The peptidomimetic macrocycle of item 8, wherein at least one ofsaid two amino acids is an α,α-disubstituted amino acid.

Item 10. The peptidomimetic macrocycle of item 8, wherein thepeptidomimetic macrocycle has the formula:

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₈, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v and w are independently integers from 1-1000;

u, x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

Item 11. The peptidomimetic macrocycle of item 1, wherein thepeptidomimetic macrocycle comprises a crosslinker linking a backboneamino group of a first amino acid to a second amino acid within thepeptidomimetic macrocycle.

Item 12. The peptidomimetic macrocycle of item 11, wherein thepeptidomimetic macrocycle has the formula (II) or (IIa):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

v and w are independently integers from 1-1000;

u, x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

Item 13. A method of increasing the circulating level of growth hormone(GH) in a subject comprising administering to the subject apeptidomimetic macrocycle of item 1.

Item 14. A method of increasing lean muscle mass in a subject comprisingadministering to the subject a peptidomimetic macrocycle of item 1.

Item 15. A method of reducing adipose tissue in a subject comprisingadministering to the subject a peptidomimetic macrocycle of item 1.

Item 16. A method of treating muscle wasting diseases, includinganorexias, cachexias (such as cancer cachexia, chronic heart failurecachexia, chronic obstructive pulmonary disease cachexia, rheumatoidarthritis cachexia) or sarcopenias in a subject comprising administeringto the subject a peptidomimetic macrocycle of item 1.

Item 17. A method of treating lipodystrophies, including HIVlipodystrophy, in a subject comprising administering to the subject apeptidomimetic macrocycle of item 1.

Item 18. A method of treating growth hormone disorders, including adultgrowth hormone deficiency and pediatric growth hormone deficiency, in asubject comprising administering to the subject a peptidomimeticmacrocycle of item 1.

Item 19. A method of treating gastroparesis or short bowel syndrome in asubject comprising administering to the subject a peptidomimeticmacrocycle of item 1.

Item 20. A method of treating muscle wasting diseases, lipodystrophies,growth hormone disorders or gastroparesis/short bowel syndrome in asubject by administering an agonist of the GHRH receptor, wherein theagonist is administered no more frequently than once daily, no morefrequently than every other day, no more frequently than twice weekly,no more frequently than weekly, or no more frequently than every otherweek.

Item 21. A method of treating muscle wasting diseases, lipodystrophies,growth hormone disorders or gastroparesis/short bowel syndrome in asubject by administering a GHRH analog, wherein the GHRH analog isadministered no more frequently than once daily, no more frequently thanevery other day, no more frequently than twice weekly, no morefrequently than weekly, or no more frequently than every other week.

Item 22. A method of increasing the circulating level of growth hormone(GH) in a subject by administering an agonist of the GHRH receptor,wherein the agonist is administered no more frequently than once daily,no more frequently than every other day, no more frequently than twiceweekly, no more frequently than weekly, or no more frequently than everyother week.

Item 23. A method of increasing the circulating level of growth hormone(GH) in a subject by administering a GHRH analog, wherein the GHRHanalog is administered no more frequently than once daily, no morefrequently than every other day, no more frequently than twice weekly,no more frequently than weekly, or no more frequently than every otherweek.

Item 24. The peptidomimetic macrocycle of item 10, wherein L₁ and L₂ areindependently alkylene, alkenylene or alkynylene.

Item 25. The peptidomimetic macrocycle of item 24, wherein L₁ and L₂ areindependently C₃-C₁₀ alkylene or alkenylene

Item 26. The peptidomimetic macrocycle of item 24, wherein L₁ and L₂ areindependently C₃-C₆ alkylene or alkenylene.

Item 27. The peptidomimetic macrocycle of item 10, wherein R₁ and R₂ areH.

Item 28. The peptidomimetic macrocycle of item 10, wherein R₁ and R₂ areindependently alkyl.

Item 29. The peptidomimetic macrocycle of item 10, wherein R₁ and R₂ aremethyl.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1: Peptidomimetic Macrocycles of the Invention

Peptidomimetic macrocycles were synthesized, purified and analyzed aspreviously described and as described below (Schafmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); andU.S. Pat. No. 7,192,713). Peptidomimetic macrocycles were designed byreplacing two or more naturally occurring amino acids with thecorresponding synthetic amino acids. Substitutions were made at i andi+4, and i and i+7 positions. Peptide synthesis was performed eithermanually or on an automated peptide synthesizer (Applied Biosystems,model 433A), using solid phase conditions, rink amide AM resin(Novabiochem), and Fmoc main-chain protecting group chemistry. For thecoupling of natural Fmoc-protected amino acids (Novabiochem), 10equivalents of amino acid and a 1:1:2 molar ratio of coupling reagentsHBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4equiv) were coupled with a 1:1:2 molar ratio of HATU (AppliedBiosystems)/HOBt/DIEA. The N-termini of the synthetic peptides wereacetylated, while the C-termini were amidated.

Purification of cross-linked compounds was achieved by high performanceliquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18column (Varian) to yield the pure compounds. Chemical composition of thepure products was confirmed by LC/MS mass spectrometry (Micromass LCTinterfaced with Agilent 1100 HPLC system) and amino acid analysis(Applied Biosystems, model 420A).

Table 4 shows a list of peptidomimetic macrocycles of the inventionprepared.

TABLE 4 (SEQ ID NOS 89-180, respectively, in order of appearance)

Olefin Exact Calc'd Obsv'd SP# Isomer mass (M + 3)/3 (M + 3)/3 SP-13410   1137.67 1137.42 SP-2 3381.99 1128.34 1127.8  SP-3 3410   1137.671137.05 SP-4 3381.99 1128.34 1127.8  SP-5 3396.01 1133.01 1132.86 SP-63525.06 1176.03 1175.76 SP-7 3493.11 1165.38 1165.02 SP-8 3480.041161.02 1160.66 SP-9 3425.98 1143   1142.53 SP-10 iso2 3425.98 1143  1142.6 SP-11 3352.94 1118.65 1118.25 SP-12 3324.93 1109.32 1108.93 SP-133436.05 1146.36 1146.15 SP-14 iso2 3436.05 1146.36 1146.08 SP-15 3422.981142   1141.94 SP-16 iso2 3422.98 1142   1141.79 SP-17 3368.92 1123.981123.66 SP-18 iso2 3368.92 1123.98 1123.73 SP-19 3338.92 1113.98 1113.37SP-20 3310.92 1101.65 1101.31 SP-21 3395.95 1132.99 1133.64 SP-223367.94 1123.65 1123.36 SP-23 3466.06 1156.36 1156.14 SP-24 3438.051147.02 1146.75 SP-25 3394.03 1132.35 1132.02 SP-26 iso2 3394.03 1132.351132.09 SP-27 3422.03 1141.68 1141.42 SP-28 iso2 3422.03 1141.68 1141.42SP-29 3414.99 1139.34 1139.05 SP-30 iso2 3414.99 1139.34 1139.05 SP-313430.95 1144.66 1144.45 SP-32 iso2 3430.95 1144.66 1145.3  SP-33 3409.991137.67 1137.42 SP-34 3324.93 1109.32 1110.29 SP-35 3352.94 1118.651119.73 SP-36 3324.93 1109.32 1110.2  SP-37 3267.88 1090.3  1091.14SP-38 3381.96 1128.33 1129.16 SP-39 3409.96 1137.66 1138.5  SP-403381.96 1128.33 1129.16 SP-41 3381.96 1128.33 1129.16 SP-42 3381.971128.33 1129.16 SP-43 3253.9  1085.64 1086.52

Exact Calc'd Obsv'd mass (M + 3)/3 (M + 3)/3 SP-50 3401.92 1134.981135.64 SP-51 3401.92 1134.98 1135.73 SP-52 3429.93 1144.32 1145.07SP-53 3401.92 1134.98 1136.19 SP-54 3273.86 1092.29 1093.18 SP-553358.91 1120.64 1121.76 SP-56 3358.91 1120.64 1121.76 SP-57 3386.921129.98 1131.1  SP-58 3358.91 1120.64 1121.76 SP-59 3230.86 1077.961079.12 SP-60 3230.82 1077.95 1079.02 SP-61 3386.96 1129.99 1130.83SP-62 3358.93 1120.65 1121.48 SP-63 3410.98 1138  1138.08 SP-64 3412.951138.66 1138.73 SP-65 3411.97 1138.33 1138.45 SP-66 3411.97 1138.331138.36

In the sequences shown above and elsewhere, the following abbreviationsare used: amino acids represented as “$” are alpha-MeS5-pentenyl-alanine olefin amino acids connected by an all-carbon i toi+4 crosslinker comprising one double bond. “%” are alpha-MeS5-pentenyl-alanine olefin amino acids connected by an all-carbon i toi+4 crosslinker comprising no double bonds (fully saturated alkylenecrosslinker). Amino acids represented as “$r8” are alpha-MeR8-octenyl-alanine olefin amino acids connected by an all-carbon i toi+7 crosslinker comprising one double bond. Amino acids represented as“% r8” are alpha-Me R8-octenyl-alanine olefin amino acids connected byan all-carbon i to i+7 crosslinker comprising no double bonds (fullysaturated alkylene crosslinker). The designation “iso1” or “iso2”indicates that the peptidomimetic macrocycle is a single isomer. Aminoacids designated as lower case “a” represent D-Alanine.

Amino acids which are used in the formation of triazole crosslinkers arerepresented according to the legend indicated below. Stereochemistry atthe alpha position of each amino acid is S unless otherwise indicated.For azide amino acids, the number of carbon atoms indicated refers tothe number of methylene units between the alpha carbon and the terminalazide. For alkyne amino acids, the number of carbon atoms indicated isthe number of methylene units between the alpha position and thetriazole moiety plus the two carbon atoms within the triazole groupderived from the alkyne.

$5a5 Alpha-Me alkyne 1,5 triazole (5 carbon)

$5n3 Alpha-Me azide 1,5 triazole (3 carbon)

$4rn6 Alpha-Me R-azide 1,4 triazole (6 carbon)

$4a5 Alpha-Me alkyne 1,4 triazole (5 carbon)

Exemplary structures of several peptidomimetic macrocycles are shown inTable 5.

TABLE 5 (SEQ ID NOS 89 and 133-135, respectively, in order ofappearance) SP# Structure SP- 1

Chemical Formula: C₁₆₁H₂₆₄N₄₂O₃₉ Exact Mass: 3410.00 Molecular Weight:3412.08 SP- 45

Chemical Formula: C₁₆₁H₂₆₈N₄₂O₃₉ Exact Mass: 3414.03 Molecular Weight:3416.11 SP- 46

Chemical Formula: C₁₆₁H₂₆₂N₄₈O₃₉ Exact Mass: 3492.00 Molecular Weight:3494.10 SP- 47

Chemical Formula: C₁₆₁H₂₆₂N₄₈O₃₉ Exact Mass: 3492.00 Molecular Weight:3494.10

Example 2: Metabolism by Purified Protease

Linear peptides and cross-linked peptidomimetic macrocycles were testedfor stability to proteolysis by Trypsin (MP Biomedicals, Solon Ohio) bysolubilizing each peptide at 10 μM concentration in 200 μL 100 mM NH4OAc(pH 7.5). The reaction was initiated by adding 3.5 μl of Trypsin (12.5μg protease per 500 μL reaction) and shaking continually in sealed vialswhile incubating in a Room Temperature (22±2° C.). The enzyme/substrateratio was 1:102 (w/w). After incubation times of 0, 5, 30, 60 and 135min the reaction was stopped by addition of equal volume of 0.2%trifluoroacetic acid. Then, the solution was immediately analyzed byLC-MS in positive detection mode. The reaction half-life for eachpeptide was calculated in GraphPad Prism by a non-linear fit ofuncalibrated MS response versus enzyme incubation time. Results areshown in FIGS. 1A and 1B.

Example 3: GHRHR Agonism Measured by cAMP

GHRH (1-29) and cross-linked peptidomimetic macrocycles were tested foragonism at the human GHRH receptor (hGHRHR) at various concentrations.Human 293 cells transiently or stably expressing hGHRHR were detachedfrom cell culture flasks with versene (Lifetechnologies), suspended inserum-free medium (50 k cells/assay point), and stimulated for 30 min atRT with GHRH (1-29) (Bachem) or cross-linked peptidomimetic macrocycles.cAMP was quantified using an HTRF®-based assay (CisBio) and usedaccording to the manufacturers instructions. An EC50% for each agonistwas calculated from a non-linear fit of response vs dose (GraphPadPrism). The maximum response was determined by stimulating with 10 μMGHRH (1-29). Results are shown in FIG. 3.

Example 4: Plasma PK/PD Study in Rats

Five peptidomimetic macrocycles of the invention (SP-1, SP-6, SP-8,SP-21, SP-32), as well as sermorelin, were studied to determinepharmacokinetic and pharmacodynamic parameters in rats. MaleSprague-Dawley rats (300 g, non-fasted, cannulated) were used. The studyhad three arms: IV administration, SC administration, and SCadministration (vehicle control). For experiments using sermorelin, adose level of 3 mg/kg IV/SC bolus was used (dose volume of 3 mL/kg doseand dose concentration of 1 mg/mL). The vehicle used was: 10 wt % N,N-Dimethylacetamide, 10 wt % DMSO, 2 wt % Solutol HS 15 in water forinjection containing 45 mg/mL (4.5 wt %) Mannitol and 25 mM (0.38 wt %)Histidine (pH 7.5; 320 mOsm/kg). The peptide was first dissolved at highconcentration in DMA and DMSO before a second dilution in Solutolvehicle.

For experiments using peptidomimetic macrocycles, 0.1 mL of DMA and 0.1mL of DMSO were used to combine with each mg of macrocycle (˜4.3-4.5 mgof macrocycle used in each experiment). Sonication was used to ensurecomplete solubilization. 0.8 mL of Solutol vehicle was used for each mgof macrocycle in DMA/DMSO. The solutions were mixed gently with pipet orlight vortexing. Fresh vials were used for each day of dosing, andmacrocycles were stored solid at −20° C. prior to formulation.

For each study arm, 2 rats were bled (350 μL) at specific timepoints (5min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h) and a 150 μLbleed was performed just before dosing. Plasma was prepared into K2EDTAtubes by centrifuging for 20 minutes at 4° C. at 2000 G maximum 30minutes after collection. From each 350 μL bleed, 120 μL weretransferred to one tube for PK studies and 50 μL to another tube for PDstudies and frozen immediately. From the 150 μL bleed, 70 μL weretransferred to one tube for PD studies and frozen immediately.

Results are shown in FIGS. 4-11.

What is claimed is:
 1. A method of treating a condition in a subject inneed thereof, the method comprising administering to the subject atherapeutically-effective amount of a peptidomimetic macrocycle, whereinthe peptidomimetic macrocycle comprises an amino acid sequence which isat least about 60% identical to GHRH 1-29, wherein the peptidomimeticmacrocycle comprises at least two macrocycle-forming linkers, whereinthe first of the at least two macrocycle-forming linkers connects afirst amino acid to a second amino acid, and the second of the at leasttwo macrocycle-forming linkers connects a third amino acid to a fourthamino acid, wherein the peptidomimetic macrocycle comprises anα,α-disubstituted amino acid, wherein the peptidomimetic macrocyclecomprises a helix.
 2. The method of claim 1, wherein the amino acidsequence of the peptidomimetic macrocycle is at least about 60%identical to an amino acid sequence chosen from the group consisting ofSEQ ID NO. 2-180.
 3. The method of claim 1, wherein the amino acidsequence of said peptidomimetic macrocycle is at least about 80%identical to an amino acid sequence chosen from the group consisting ofSEQ ID NO. 2-180.
 4. The method of claim 1, wherein the amino acidsequence of said peptidomimetic macrocycle is at least about 90%identical to an amino acid sequence chosen from the group consisting ofSEQ ID NO. 2-180.
 5. The method of claim 1, wherein the amino acidsequence of said peptidomimetic macrocycle is chosen from the groupconsisting of SEQ ID NO. 2-180.
 6. The method of claim 1, wherein thepeptidomimetic macrocycle comprises an α-helix.
 7. The method of claim1, wherein the condition is a cachexia.
 8. The method of claim 1,wherein the condition is a lipodystrophy.
 9. The method of claim 1,wherein the condition is a growth hormone disorder.
 10. The method ofclaim 1, wherein each of the at least two macrocycle-forming linkersconnects a pair of amino acids corresponding to one of the followinglocations of amino acids: 4 and 8; 5 and 12; 8 and 12; 8 and 15; 9 and16; 12 and 16; 12 and 19; 15 and 22; 18 and 25; 21 and 25; 21 and 28; 22and 29; or 25 and 29 of GHRH 1-29.
 11. The method of claim 1, whereineach of the at least two macrocycle-forming linkers connects a pair ofamino acids corresponding to one of the following locations of aminoacids: 4 and 8; 5 and 12; 12 and 19; 15 and 22; 18 and 25; 21 and 25; or21 and 28 of GHRH 1-29.
 12. The method of claim 1, wherein the firstmacrocycle-forming linker connects a pair of amino acids correspondingto one of the following locations of amino acids: 4 and 8; 5 and 12; 8and 12; 8 and 15; 9 and 16; 12 and 16; or 12 and 19 of GHRH 1-29; andthe second macrocycle-forming linker connects a pair of amino acidscorresponding to one of the following locations of amino acid 15 and 22;18 and 25; 21 and 25; 21 and 28; 22 and 29; or 25 and 29 of GHRH 1-29.13. The method of claim 1, wherein the first macrocycle-forming linkerconnects a pair of amino acids corresponding to one of the followinglocations of amino acids: 4 and 8; 5 and 12; 8 and 12; or 12 and 19 ofGHRH 1-29; and the second macrocycle-forming linker connects a pair ofamino acids corresponding to one of the following locations of aminoacid 15 and 22; 18 and 25; 21 and 25; or 21 and 28 of GHRH 1-29.
 14. Themethod of claim 1, wherein the first macrocycle-forming linker connectsa pair of amino acids corresponding to amino acids locations 4 and 8 ofGHRH 1-29.
 15. The method of claim 1, wherein the secondmacrocycle-forming linker connects a pair of amino acids correspondingto amino acid locations 21 and 25 of GHRH 1-29.
 16. The method of claim1, wherein the first macrocycle-forming linker connects a pair of aminoacids corresponding to amino acid locations 4 and 8 of GHRH 1-29; andthe second macrocycle-forming linker connects a pair of amino acidscorresponding to amino acid locations 21 and 25 of GHRH 1-29.
 17. Themethod of claim 1, wherein each amino acid connected by the at least twomacrocycle-forming linkers is an α,α-disubstituted amino acid.
 18. Themethod of claim 1, wherein the peptidomimetic macrocycle has theformula:

wherein: each A, C, D, and E is independently an amino acid; B is anamino acid or

L is a macrocycle-forming linker of the formula -L₁-L₂-; and wherein A,B, C, D, and E, taken together with the crosslinked amino acidsconnected by the macrocycle-forming linker L, form the amino acidsequence of the peptidomimetic macrocycle; R₁ and R₂ are independently—H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl, unsubstituted or substituted withhalo-; R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅; L₁ and L₂ areindependently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₄—K—R₄-]_(n), each being optionally substituted with R₅; each R₄ isalkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene; each K is O, S, SO, SO₂,CO, CO₂, or CONR₃; each R₅ is independently halogen, alkyl, —OR₆,—N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, a fluorescent moiety, aradioisotope or a therapeutic agent; each R₆ is independently —H, alkyl,alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, afluorescent moiety, a radioisotope or a therapeutic agent; R₇ is —H,alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue; R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅, or part of a cyclicstructure with an E residue; v and w are independently integers from1-100; u is an integer from 1 to 3; x, y and z are independentlyintegers from 0-10; and n is an integer from 1-5.
 19. The method ofclaim 18, wherein L₁ and L₂ are independently alkylene, alkenylene oralkynylene.
 20. The method of claim 18, wherein L₁ and L₂ areindependently C₃-C₁₀ alkylene or alkenylene.
 21. The method of claim 18,wherein L₁ and L₂ are independently C₃-C₆ alkylene or alkenylene. 22.The method of claim 18, wherein R₁ and R₂ are independently H.
 23. Themethod of claim 18, wherein R₁ and R₂ are independently alkyl.
 24. Themethod of claim 18, wherein R₁ and R₂ are independently methyl.
 25. Themethod of claim 18, wherein one of R₁ and R₂ is —H, and the other of R₁and R₂ is alkyl.
 26. The method of claim 18, wherein the sum of x+y+z is2, 3, or
 6. 27. The method of claim 18, wherein each of v and w isindependently an integer from 1 to
 25. 28. The method of claim 18,wherein A, B, C, D, and E, taken together with the crosslinked aminoacids connected by the macrocycle-forming linker L, form an amino acidsequence which is at least about 60% identical to GHRH 1-29.
 29. Themethod of claim 18, wherein u is
 2. 30. The method of claim 29, whereinthe peptidomimetic macrocycle has the formula:

wherein each A, C, D, and E is independently an amino acid; B is anamino acid or

L′ is a macrocycle-forming linker of the formula -L₁′-L₂′-; and whereinA, B, C, D, and E, taken together with the crosslinked amino acidsconnected by the macrocycle-forming linkers L and L′, form the aminoacid sequence of the peptidomimetic macrocycle; R₁′ and R₂′ areindependently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-; L₁′ and L₂′ are independently alkylene,alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₄—K—R₄-]_(n), each being optionally substituted with R₅; each K isindependently O, S, SO, SO₂, CO, CO₂, or CONR₃; R₇′ is —H, alkyl,alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substitutedwith R₅, or part of a cyclic structure with a D residue; R₈′ is —H,alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue; v′ and w′ are independently integers from 1-100; x′, y′ and z′are independently integers from 0-10; and n is an integer from 1-5. 31.The method of claim 30, wherein the sum of x′+y′+z′ is 2, 3, or
 6. 32.The method of claim 1, wherein the condition is a muscle wastingdisease.
 33. The method of claim 1, wherein the condition is a growthhormone deficiency.
 34. The method of claim 1, wherein the condition isgastroparesis.
 35. A method of treating a condition in a subject in needthereof, the method comprising administering to the subject atherapeutically-effective amount of a peptidomimetic macrocycle, whereinthe peptidomimetic macrocycle comprises an amino acid sequence offormula:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29  (SEQID NO: 1) wherein: X1 is Tyr or His; X2 is Ala, D-Ala, or Val; X3 isAsp; X4 is Ala or a crosslinked amino acid; X5 is Ile; X6 is Phe; X7 isThr; X8 is Gln, Asn, or a crosslinked amino acid; X9 is Ser or acrosslinked amino acid; X10 is Tyr; X11 is Arg, Ala or Gln; X12 is Lys,Ala, Gln or a crosslinked amino acid; X13 is Val or Ile; X14 is Leu; X15is Gly, Ala or a crosslinked amino acid; X16 is Gln, Glu or acrosslinked amino acid; X17 is Leu; X18 is Ser, Tyr or a crosslinkedamino acid; X19 is Ala or a crosslinked amino acid; X20 is Arg or Gln;X21 is Lys, Gln or a crosslinked amino acid; X22 is Leu, Ala, or acrosslinked amino acid; X23 is Leu; X24 is Gln, Glu or His; X25 is Asp,Glu or a crosslinked amino acid; X26 is Ile; X27 is Met, Ile, Leu orNle; X28 is Ser or a crosslinked amino acid; X29 is Arg, Ala, Gln or acrosslinked amino acid; wherein at least one of the crosslinked aminoacids is an α,α-disubstituted amino acid; wherein the peptidomimeticmacrocycle comprises at least one macrocycle-forming linker connectingat least one pair of amino acids selected from X1-X29; L is amacrocycle-forming linker of the formula -L₁-L₂-; L₁ and L₂ areindependently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₄—K—R₄-]_(n), each being optionally substituted with R₅, wherein n isan integer from 1-5; each R₄ is alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene; each K is O, S, SO, SO₂, CO, or CO₂; each R₅ isindependently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR_(E), —SO₂R₆,—CO₂R₆, a fluorescent moiety, a radioisotope or a therapeutic agent; andeach R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent.
 36. The method of claim 35, wherein the at leastone macrocycle-forming linker connects one of the following pairs ofamino acids: X4 and X8; X5 and X12; X8 and X12; X8 and X15; X9 and X16;X12 and X16; X12 and X19; X15 and X22; X18 and X25; X21 and X25; X21 andX28; X22 and X29; or X25 and X29.
 37. The method of claim 35, whereinthe at least one macrocycle-forming linker connects one of the followingpairs of amino acids: X4 and X8; X5 and X12; X12 and X19; X15 and X22;X18 and X25; X21 and X25; or X21 and X28.
 38. The method of claim 35,wherein the condition is a muscle wasting disease.
 39. The method ofclaim 35, wherein the condition is a growth hormone deficiency.
 40. Themethod of claim 35, wherein the condition is gastroparesis.
 41. Themethod of claim 35, wherein the condition is a cachexia.
 42. The methodof claim 35, wherein the condition is a lipodystrophy.
 43. The method ofclaim 35, wherein the condition is a growth hormone disorder.